Polymeric BODIPY dyes and methods for using the same

ABSTRACT

Polymeric BODIPY dyes including light harvesting BODIPY unit-comprising multichromophores are provided. In some embodiments, the dyes are polymeric tandem dyes that include a light harvesting BODIPY unit-comprising multichromophore and an acceptor chromophore covalently linked to the multichromophore in energy-receiving proximity therewith. The polymeric tandem dyes may be covalently linked to a specific binding member. Also provided are methods of evaluating a sample for the presence of a target analyte and methods of labelling a target molecule using compositions including the polymeric tandem dyes. Kits and systems for practicing the subject methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 62/132,446,filed Mar. 12, 2015, the disclosure of which application is incorporatedherein by reference.

INTRODUCTION

Fluorescent dyes are compounds which, when irradiated with light of awavelength which they absorb, emit light of a (usually) differentwavelength. Fluorescent dyes find use in a variety of applications inbiochemistry, biology and medicine, e.g. in diagnostic kits, inmicroscopy or in drug screening. Fluorescent dyes are characterized by anumber of parameters allowing a user to select a suitable dye dependingon the desired purpose. Parameters of interest include the excitationwavelength maximum, the emission wavelength maximum, the Stokes shift,the extinction coefficient, the fluorescence quantum yield and thefluorescence lifetime. Dyes may be selected according to the applicationof interest in order to, e.g., allow penetration of exciting radiationinto biological samples, to minimize background fluorescence and/or toachieve a high signal-to-noise ratio.

Molecular recognition involves the specific binding of two molecules.Molecules which have binding specificity for a target biomolecule finduse in a variety of research and diagnostic applications, such as thelabelling and separation of analytes, flow cytometry, in situhybridization, enzyme-linked immunosorbent assays (ELISAs), western blotanalysis, magnetic cell separations and chromatography. Targetbiomolecules may be detected by labelling with a fluorescent dye.

SUMMARY

Polymeric BODIPY dyes including light harvesting BODIPY unit-comprisingmultichromophores are provided. In some embodiments, the dyes arepolymeric tandem dyes that include a light harvesting BODIPYunit-comprising multichromophore and an acceptor chromophore covalentlylinked to the multichromophore in energy-receiving proximity therewith.The polymeric tandem dyes may be covalently linked to a specific bindingmember. Also provided are methods of evaluating a sample for thepresence of a target analyte and methods of labelling a target moleculeusing compositions including the polymeric tandem dyes. Kits and systemsfor practicing the subject methods are also provided.

BRIEF DESCRIPTION OF THE FIGURES

It is understood that the drawings, described below, are forillustration purposes only. The drawings are not intended to limit thescope of the present teachings in any way.

FIG. 1 illustrates absorption and emission of exemplary polymeric tandemdyes with a variety of acceptor dyes attached at the internal linkersite. No specific binding member is attached for these structures.

FIG. 2 illustrates absorption and emission of exemplary polymeric tandemdyes with a variety of dye molecules attached at the internal linkersite. Absorption for all solutions is 0.04 OD. Note that the emissionintensity is significantly higher for polymers with an acceptorchromophore attached relative to the polymer alone. No specific bindingmember is attached to these polymers.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a primer”refers to one or more primers, i.e., a single primer and multipleprimers. It is further noted that the claims can be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

As used herein, the term “sample” relates to a material or mixture ofmaterials, in some cases in liquid form, containing one or more analytesof interest. In some embodiments, the term as used in its broadestsense, refers to any plant, animal or bacterial material containingcells or producing cellular metabolites, such as, for example, tissue orfluid isolated from an individual (including without limitation plasma,serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) orfrom in vitro cell culture constituents, as well as samples from theenvironment. The term “sample” may also refer to a “biological sample”.As used herein, the term “a biological sample” refers to a wholeorganism or a subset of its tissues, cells or component parts (e.g. bodyfluids, including, but not limited to, blood, mucus, lymphatic fluid,synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amnioticcord blood, urine, vaginal fluid and semen). A “biological sample” canalso refer to a homogenate, lysate or extract prepared from a wholeorganism or a subset of its tissues, cells or component parts, or afraction or portion thereof, including but not limited to, plasma,serum, spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors and organs. In certain embodiments, the sample hasbeen removed from an animal or plant. Biological samples may includecells. The term “cells” is used in its conventional sense to refer tothe basic structural unit of living organisms, both eukaryotic andprokaryotic, having at least a nucleus and a cell membrane. In certainembodiments, cells include prokaryotic cells, such as from bacteria. Inother embodiments, cells include eukaryotic cells, such as cellsobtained from biological samples from animals, plants or fungi.

As used herein, the terms “affinity” and “avidity” have the same meaningand may be used interchangeably herein. “Affinity” refers to thestrength of binding, increased binding affinity being correlated with alower Kd.

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

As used herein, the terms “support bound” and “linked to a support” areused interchangeably and refer to a moiety (e.g., a specific bindingmember) that is linked covalently or non-covalently to a support ofinterest. Covalent linking may involve the chemical reaction of twocompatible functional groups (e.g., two chemoselective functionalgroups, an electrophile and a nucleophile, etc.) to form a covalent bondbetween the two moieties of interest (e.g. a support and a specificbinding member). In some cases, non-covalent linking may involvespecific binding between two moieties of interest (e.g., two affinitymoieties such as a hapten and an antibody or a biotin moiety and astreptavidin, etc.). In certain cases, non-covalent linking may involveabsorption to a substrate.

As used herein, the term “biomolecule” refers to an organic molecule ormacromolecule of a naturally occurring class of molecules, or aderivative thereof. Biomolecule is meant to encompass polypeptides(e.g., a peptide, an antibody or an antibody fragment), polynucleotides,carbohydrates (e.g., sugars) and lipids. In some cases, the biomoleculeis a specific binding member (e.g., as described herein).

As used herein, the term “polypeptide” refers to a polymeric form ofamino acids of any length, including peptides that range from 2-50 aminoacids in length and polypeptides that are greater than 50 amino acids inlength. The terms “polypeptide” and “protein” are used interchangeablyherein. The term “polypeptide” includes polymers of coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones in which theconventional backbone has been replaced with non-naturally occurring orsynthetic backbones. A polypeptide may be of any convenient length,e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or moreamino acids, 20 or more amino acids, 50 or more amino acids, 100 or moreamino acids, 300 or more amino acids, such as up to 500 or 1000 or moreamino acids. “Peptides” may be 2 or more amino acids, such as 4 or moreamino acids, 10 or more amino acids, 20 or more amino acids, such as upto 50 amino acids. In some embodiments, peptides are between 5 and 30amino acids in length.

As used herein, the term “isolated,” refers to an moiety of interestthat is at least 60% free, at least 75% free, at least 90% free, atleast 95% free, at least 98% free, and even at least 99% free from othercomponents with which the moiety is associated with prior topurification.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have 10 or more, such as 100 or more, 1000 or more, 10,000 or more,100,000 or more, 10⁶ or more, 10⁷ or more, 10⁸ or more or 10⁹ or moremembers.

Numeric ranges are inclusive of the numbers defining the range.

The term “separating”, as used herein, refers to physical separation oftwo elements (e.g., by size or affinity, etc.) as well as degradation ofone element, leaving the other intact.

As used herein, the term “specific binding” refers to the ability of acapture agent (or a first member of a specific binding pair) topreferentially bind to a particular analyte (or a second member of aspecific binding pair) that is present, e.g., in a homogeneous mixtureof different analytes. In some instances, a specific binding interactionwill discriminate between desirable and undesirable analytes in a samplewith a specificity of 10-fold or more for a desirable analyte over anundesirable analytes, such as 100-fold or more, or 1000-fold or more. Insome cases, the affinity between a capture agent and analyte when theyare specifically bound in a capture agent/analyte complex is at least10⁻⁸M, at least 10⁻⁹M, such as up to 10⁻¹⁰M.

The methods described herein include multiple steps. Each step may beperformed after a predetermined amount of time has elapsed betweensteps, as desired. As such, the time between performing each step may be1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds ormore, 5 minutes or more, 10 minutes or more, 60 minutes or more andincluding 5 hours or more. In certain embodiments, each subsequent stepis performed immediately after completion of the previous step. In otherembodiments, a step may be performed after an incubation or waiting timeafter completion of the previous step, e.g., a few minutes to anovernight waiting time.

As used herein, the term “linker” or “linkage” refers to a linkingmoiety that connects two groups and has a backbone of 100 atoms or lessin length. A linker or linkage may be a covalent bond that connects twogroups or a chain of between 1 and 100 atoms in length, for example achain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbonatoms in length, where the linker may be linear, branched, cyclic or asingle atom. In some cases, the linker is a branching linker that refersto a linking moiety that connects three or more groups. In certaincases, one, two, three, four or five or more carbon atoms of a linkerbackbone may be optionally substituted with a sulfur, nitrogen or oxygenheteroatom. The bonds between backbone atoms may be saturated orunsaturated, and in some cases not more than one, two, or threeunsaturated bonds are present in a linker backbone. The linker mayinclude one or more substituent groups, for example with an alkyl, arylor alkenyl group. A linker may include, without limitations,polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, whichmay be straight or branched, e.g., methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl(t-butyl), and the like. The linker backbone may include a cyclic group,for example, an aryl, a heterocycle or a cycloalkyl group, where 2 ormore atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included inthe backbone. A linker may be cleavable or non-cleavable.

As used herein, the terms “polyethylene oxide”, “PEO”, “polyethyleneglycol”, “PEG” and “PEG moiety” are used interchangeably and refer to apolymeric group including a chain described by the formula—(CH₂—CH₂—O—)_(n)— or a derivative thereof. In some embodiments, “n” is5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 orless, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, suchas 3 to 15, or 10 to 15. It is understood that the PEG polymeric groupmay be of any convenient length and may include a variety of terminalgroups and/or further substituent groups, including but not limited to,alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amido terminal and/orsubstituent groups. PEG groups that may be adapted for use in thesubject multichromophores include those PEGs described by S. Zalipsky in“Functionalized poly(ethylene glycol) for preparation of biologicallyrelevant conjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165; andby Zhu et al in “Water-Soluble Conjugated Polymers for Imaging,Diagnosis, and Therapy”, Chem. Rev., 2012, 112 (8), pp 4687-4735.

As used herein, the terms “chemoselective functional group”,“chemoselective tag” and “conjugation tag” are used interchangeably andrefer to a functional group that can selectively react with anothercompatible functional group to form a covalent bond, in some cases,after optional activation of one of the functional groups.Chemoselective functional groups of interest include, but are notlimited to, thiols and maleimide or iodoacetamide, amines and carboxylicacids or active esters thereof, as well as groups that can react withone another via Click chemistry, e.g., azide and alkyne groups (e.g.,cyclooctyne groups), as well as hydroxyl, hydrazido, hydrazino,aldehyde, ketone, azido, alkyne, phosphine, epoxide, and the like. Insome cases, the chemoselective functional group is a protectedfunctional group that must be deprotected prior to covalent linking. Incertain instances, the chemoselective functional group is may beactivated prior to or during covalent linking with a compatiblefunctional group.

As used herein, the term “alkyl” by itself or as part of anothersubstituent refers to a saturated branched or straight-chain monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. Alkyl groups of interest include,but are not limited to, methyl; ethyl, propyls such as propan-1-yl orpropan-2-yl; and butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl or 2-methyl-propan-2-yl. In some embodiments, analkyl group includes from 1 to 20 carbon atoms. In some embodiments, analkyl group includes from 1 to 10 carbon atoms. In certain embodiments,an alkyl group includes from 1 to 6 carbon atoms, such as from 1 to 4carbon atoms. This term includes, by way of example, linear and branchedhydrocarbyl groups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl(CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—),n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of an aromatic ring system. Arylgroups of interest include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene and the like. In certain embodiments, an aryl groupincludes from 6 to 20 carbon atoms. In certain embodiments, an arylgroup includes from 6 to 12 carbon atoms. Examples of an aryl group arephenyl and naphthyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a heteroaromatic ring system. Heteroarylgroups of interest include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene,triazole, xanthene, benzodioxole and the like. In certain embodiments,the heteroaryl group is from 5-20 membered heteroaryl. In certainembodiments, the heteroaryl group is from 5-10 membered heteroaryl. Incertain embodiments, heteroaryl groups are those derived from thiophene,pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline,imidazole, oxazole and pyrazine.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl andsubstituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Substituents of interest include, but are not limited to, alkylenedioxy(such as methylenedioxy), -M, —R⁶⁰, —O⁻, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R⁶⁰, —OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰),(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹,—C(O)O⁻, —C(S) OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹,—NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹,R⁶² and R⁶³ are independently hydrogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independentlyhydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, heteroaryl or substituted heteroaryl, or optionallyR⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certainembodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R⁶⁰,—OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹),—C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —NR⁶²C(O)NR⁶⁰R⁶¹.In certain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O⁻),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻. Incertain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(O)OR⁶⁰, —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above. Forexample, a substituted group may bear a methylenedioxy substituent orone, two, or three substituents selected from a halogen atom, a(1-4C)alkyl group and a (1-4C)alkoxy group. When the group beingsubstituted is an aryl or heteroaryl group, the substituent(s) (e.g., asdescribed herein) may be referred to as “aryl substituent(s)”.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

As summarized above, polymeric tandem dyes are provided. In someembodiments, the polymeric tandem dyes include a light harvesting BODIPYunit-comprising multichromophore and an acceptor chromophore covalentlylinked to the multichromophore in energy-receiving proximity therewith.The polymeric tandem dyes may be covalently linked to a specific bindingmember. Also provided are methods of evaluating a sample for thepresence of a target analyte and methods of labelling a target moleculeusing compositions including the polymeric tandem dyes. Kits and systemsfor practicing the subject methods are also provided.

Before the various embodiments are described in greater detail, it is tobe understood that the teachings of this disclosure are not limited tothe particular embodiments described, and as such can, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present teachings will be limitedonly by the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

In further describing the subject invention, polymeric dyes and tandemdyes including an acceptor chromophore are described first in greaterdetail. Next, conjugates which include the polymeric dyes are described.Then, methods of interest in which compositions including the subjectpolymeric tandem dyes find use are reviewed. Systems and kits that maybe used in practicing methods of the invention are also described.

Light Harvesting BODIPY Unit-Comprising Multichromophores

As summarized above, the present disclosure provides a polymeric BODIPYdye. In some embodiments, the polymeric dye includes a light harvestingBODIPY unit-comprising multichromophore. In some embodiments, themultichromophore is itself fluorescent. In certain instances, themultichromophore is a polymeric tandem dye. As such, in someembodiments, the multichromophore further includes an acceptorchromophore covalently linked to the multichromophore inenergy-receiving proximity therewith.

As used herein the terms, “light harvesting multichromophore”,“polymeric dye” and “conjugated polymer” are used interchangeably andrefer to a conjugated polymer which has a structure capable ofharvesting light with a particular absorption maximum wavelength andconverting it to emitted light at a longer emission maximum wavelength.In some cases, the light harvesting multichromophore is itselffluorescent. Conjugated polymers (CPs) are characterized by adelocalized electronic structure and may have an effective conjugationlength that is substantially shorter than the length of the polymerchain, because the backbone may contain a large number of conjugatedsegments in close proximity. In some cases, conjugated polymers areefficient for light harvesting and provide for optical amplification viaForster energy transfer to an acceptor. In some embodiments, theconjugated polymer includes a plurality of first optically active unitsforming a conjugated system, having an absorption wavelength (e.g., asdescribed herein) at which the first optically active units absorbslight to form an excited state. In certain instances, the polymeric dyeincludes a conjugated polymer segment or an oligomeric structureincluding bandgap-lowering n-conjugated repeat units.

As used herein the term “unit” refers to a structural subunit of apolymer. The term unit is meant to include monomers, co-monomers,co-blocks, conjugated segments, repeating units, and the like. A“repeating unit” is a subunit of a polymer that is defined by theminimum number of distinct structural features that are required for theunit to be considered monomeric, such that when the unit is repeated ntimes, the resulting structure describes the polymer or a block thereof.In some cases, the polymer may include two or more different repeatingunits, e.g., when the polymer is a multiblock polymer, each block maydefine a distinct repeating unit. In some cases, a repeating unit of thepolymer includes a single monomer group. In certain instances, arepeating unit of the polymer includes two or more monomer groups, i.e.,co-monomer groups, such as two, three, four or more co-monomer groups.As used herein, the term “co-monomer” or “co-monomer group” refers to astructural unit of a polymer that may itself be part of a repeating unitof the polymer. In some embodiments, the conjugated polymer includes ablock copolymer that is composed of blocks of polymerized monomers. Insuch cases, the block copolymer may be described as having distinctrepeating units each corresponding to a distinct co-block of thepolymer. In some cases, the polymer is a diblock copolymer that containstwo different co-blocks. In such cases, the polymer may be described asincluding co-blocks, where each co-block may be composed of co-monomers,such as one, two, three or more co-monomers.

As used herein, the term “BODIPY unit” refers to a structural subunit ofthe multichromophore which includes a chromophore having the followingboron-dipyrromethene (BODIPY) core structure:

where each R is independently selected from the group consisting of F,OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl andsubstituted alkynyl. The BODIPY core structure may be linked to adjacentunits of the multichromophore via any convenient positions of the corestructure, and may be further optionally substituted. In some instances,the BODIPY unit is capable of π conjugation to adjacent units of thepolymer. In some embodiments, the BODIPY unit defines a repeating unit.In certain embodiments, the BODIPY unit defines a co-monomer which ispart of a repeating unit. Any convenient BODIPY-containing structuresmay be adapted for use in the subject multichromophores as a BODIPYunit. BODIPY-containing structures of interest include, but are notlimited to, those BODIPY dyes and derivatives described by Loudet andBurgess in “BODIPY Dyes and Their Derivatives: Syntheses andSpectroscopic Properties”, Chem. Rev. 2007, 107 (11): 4891-4932.

In certain embodiments, the BODIPY unit is described by the structure:

where:

R¹, R², R³ and R⁴ are each independently selected from H, an alkyl or asubstituted alkyl;

R⁵ is an alkyl, a substituted alkyl, an aryl, a substituted aryl, aheteroaryl, a substituted heteroaryl, wherein R⁵ is optionallysubstituted with a water solubilizing group; and

each R is selected from the group consisting of F, OH, H, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, alkynyl and substituted alkynyl.

Any convenient light harvesting multichromophores may be adapted toinclude a BODIPY unit. Light harvesting multichromophores of interestthat may be modified to include a BODIPY unit include, but are notlimited to, those dyes described by Gaylord et al. in US PublicationNos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549,20120028828, 20120252986 and 20130190193 and U.S. Pat. Nos. 8,575,303and 8,802450, the disclosures of which Publications and Patents areherein incorporated by reference in their entirety; and Gaylord et al.,J. Am. Chem. Soc., 2001, 123 (26), pp 6417-6418; Feng et al., Chem. Soc.Rev., 2010, 39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011,133 (32), pp 12600-12607, the disclosures of which are hereinincorporated by reference in their entirety.

The subject multichromophore may be water soluble. Any convenient watersolubilizing groups may be included in the multichromophore to providefor increased water-solubility of the dye. While the increase insolubility may vary, in some instances the increase (as compared to thecompound without the WSG(s)) is 2 fold or more, e.g., 5 fold, 10 fold,25 fold, 50 fold, 100 fold or more. The term “water solubilizing group”(WSG) refers to a group that is well solvated in aqueous environmentse.g., under physiological conditions, and that imparts improved watersolubility to the molecules to which it is attached. In someembodiments, a WSG increases the solubility of the multichromophore in apredominantly aqueous solution, as compared to a multichromophore whichlacks the WSG. The water soluble groups may be any convenienthydrophilic group that is well solvated in aqueous environments. In somecases, the hydrophilic water soluble group is charged, e.g., positivelyor negatively charged. In certain cases, the hydrophilic water solublegroup is a neutral hydrophilic group. In some embodiments, the WSG is ahydrophilic polymer, e.g., a polyethylene glycol, a cellulose, achitosan, or a derivative thereof. Water soluble groups of interestinclude, but are not limited to, carboxylate, phosphonate, phosphate,sulfonate, sulfate, sulfinate, sulfonium, ester, polyethylene glycols(PEG) and modified PEGs, hydroxyl, amine, ammonium, guanidinium,pyridinium, polyamine and sulfonium, polyalcohols, straight chain orcyclic saccharides, primary, secondary, tertiary, or quaternary aminesand polyamines, phosphonate groups, phosphinate groups, ascorbategroups, glycols, including, polyethers, —COOM′, —SO₃M′, —PO₃M′, —NR₃ ⁺,Y′, (CH₂CH₂O)_(p)R and mixtures thereof, where Y′ can be any halogen,sulfate, sulfonate, or oxygen containing anion, p can be 1 to 500, eachR can be independently H or an alkyl (such as methyl) and M′ can be acationic counterion or hydrogen, —(CH₂CH₂O)_(yy)CH₂CH₂XR^(yy),—(CH₂CH₂O)_(yy)CH₂CH₂X—, —X(CH₂CH₂O)_(yy)CH₂CH₂—, glycol, andpolyethylene glycol, wherein yy is selected from 1 to 1000, X isselected from O, S, and NR^(ZZ), and R^(ZZ) and R^(YY) are independentlyselected from H and C₁₋₃ alkyl.

Multiple WSGs may be included at a single location in the subjectmultichoromophores via a branching linker. Any convenient branchinglinkers may be utilized in order to provide for linking to multiple WSG.Branching linkers of interest include, but are not limited, tertiaryamino groups (e.g., where N is a branching atom), amino acid residues,substituted aryl groups, substituted heteroaryl groups, substitutedheterocyclic groups, dendrimers, and the like. In certain embodiments,the branching linker is an aralkyl substituent that is furtherdisubstituted with water soluble group(s). As such, in some cases, thebranching linker group is a substituent of the multichromophore thatconnects the multichromophore to two or more water soluble groups. Insome cases, the incorporation of multiple WSGs via branching linkersimparts a desirable solubility on the multichromophore. In someembodiments, the multichromophore includes substituent(s) selected fromthe group consisting of an alkyl, an aralkyl and a heterocyclic group,each group further substituted with a water solubilizing group. Incertain cases, the WSG is a hydrophilic polymer group, such as apolyethylglycol (PEG) (e.g., a PEG of 2-20 units).

The multichromophore may be of any convenient length. In some cases, theparticular number of monomeric repeating units or segments of themultichromophore may fall within the range of 2 to 500,000, such as 2 to100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000 units orsegments, or such as 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to100,000, or 500 to 50,000 units or segments. In some instances, theparticular number of monomeric repeating units or segments of themultichromophore may fall within the range of 2 to 1,000, such as 2 to500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to100, 9 to 100 or 10 to 100 units or segments.

The multichromophore may be of any convenient molecular weight (MW). Insome cases, the MW of the multichromophore may be expressed as anaverage molecular weight. In some instances, the polymeric dye has anaverage molecular weight of from 500 to 500,000, such as from 1,000 to100,000, from 2,000 to 100,000, from 10,000 to 100,000 or even anaverage molecular weight of from 50,000 to 100,000.

In some embodiments, the BODIPY unit constitutes 25% or more by molarityof the multichromophore, such as 30% or more, 40% or more, 45% or more,50% or more, 60% or more, 70% or more, or even more by molarity of themultichromophore. In such cases, the multichromophore may include 5 ormore repeating units, such as 10 or more, 20 or more, 30 or more, 40 ormore, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 ormore, 200 or more, 500 or more, 1000 or more, 10,000 or more, or evenmore repeating units. In such cases, the multichromophore may include 5or more co-monomer units, such as 10 or more, 20 or more, 30 or more, 40or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100or more, 200 or more, 500 or more, 1000 or more, 10,000 or more, or evenmore co-monomer units.

The subject multichromophore may have one or more desirablespectroscopic properties, such as a particular absorption maximumwavelength, a particular emission maximum wavelength, extinctioncoefficient, quantum yield, narrow band spectral features, low energyabsorption bands, and the like.

In certain embodiments, the multichromophore has narrow band spectralfeatures. A narrow band spectral feature refers to an absorbance oremission spectra with a full width at half maximum (FWHM) of 50 nm orless with peaks centered at 500 nm or more. In some embodiments, the dyehas low energy absorption bands having a bandwidth of 200 nm or less,such as 150 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less,20 nm or less, or even less. In some cases, the bandwidth is determinedvia a full width at half maximum (FWHM) measurement. In certainembodiments, the dye has low energy absorption bands having a bandwidthof 50 nm or less.

In some embodiments, the multichromophore has an absorption maximumwavelength in the range of 300 to 900 nm, such as 350 to 850 nm, 350 to600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm or400 to 500 nm, where specific examples of absorption maxima of interestinclude, but are not limited to: 590 nm, 630 nm, 650 nm, 680 nm and 750nm. In certain embodiments, the multichromophore has an absorptionmaximum wavelength of 590 nm±5 nm, 630 nm±5 nm, 650 nm±5 nm, 680 nm±5 nmor 750 nm±5 nm. In some embodiments, the multichromophore has anemission maximum wavelength in the range of 300 to 900 nm, such as 350to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm,390 to 500 nm or 400 to 500 nm, where specific examples of emissionmaxima of interest include, but are not limited to: 605 nm, 650 nm, 680nm, 700 nm and 805 nm. In certain embodiments, the multichromophore hasan emission maximum wavelength of 605 nm±5 nm, 650 nm±5 nm, 680 nm±5 nm,700 nm±5 nm or 805 nm±5 nm.

In some instances, the multichromophore has an extinction coefficient of5×10⁵ cm⁻¹M⁻¹ or more, such as 6×10⁵ cm⁻¹M⁻¹ or more, 7×10⁵ cm⁻¹M⁻¹ ormore, 8×10⁵ cm⁻¹M⁻¹ or more, 9×10⁵ cm⁻¹M⁻¹ or more, such as 1×10⁶cm⁻¹M⁻¹ or more, 1.5×10⁶ cm⁻¹M⁻¹ or more, 2×10⁶ cm⁻¹M⁻¹ or more, 2.5×10⁶cm⁻¹M⁻¹ or more, 3×10⁶ cm⁻¹M⁻¹ or more, 4×10⁶ cm⁻¹M⁻¹ or more, 5×10⁶cm⁻¹M⁻¹ or more, 6×10⁶ cm⁻¹M⁻¹ or more, 7×10⁶ cm⁻¹M⁻¹ or more, or 8×10⁶cm⁻¹M⁻¹ or more. In such cases, the multichromophore may have 5 or morerepeating units, such as 6 or more, 7 or more, 8 or more, 9 or more, 10or more, or even more repeating units. In some embodiments, themultichromophore has a molar extinction coefficient of 5×10⁵ M⁻¹ cm⁻¹ ormore. In certain embodiments, the multichromophore has a molarextinction coefficient of 1×10⁶ M⁻¹ cm⁻¹ or more.

In some instances, the multichromophore has an extinction coefficient of40,000 cm⁻¹M⁻¹ per co-monomer or more, such as 45,000 cm⁻¹M⁻¹ perco-monomer or more, 50,000 cm⁻¹M⁻¹ per co-monomer or more, 55,000cm⁻¹M⁻¹ per co-monomer or more, 60,000 cm⁻¹M⁻¹ per co-monomer or more,70,000 cm⁻¹M⁻¹ per co-monomer or more, 80,000 cm⁻¹M⁻¹ per co-monomer ormore, 90,000 cm⁻¹M⁻¹ per co-monomer or more, 100,000 cm⁻¹M⁻¹ perco-monomer or more, or even more. In some instances, themultichromophore has an extinction coefficient of 40,000 cm⁻¹M⁻¹ perrepeating unit or more, such as 45,000 cm⁻¹M⁻¹ per repeating unit ormore, 50,000 cm⁻¹M⁻¹ per repeating unit or more, 55,000 cm⁻¹M⁻¹ perrepeating unit or more, 60,000 cm⁻¹M⁻¹ per repeating unit or more,70,000 cm⁻¹M⁻¹ per repeating unit or more, 80,000 cm⁻¹M⁻¹ per repeatingunit or more, 90,000 cm⁻¹M⁻¹ per repeating unit or more, 100,000 cm⁻¹M⁻¹per repeating unit or more, 100,000 cm⁻¹M⁻¹ per repeating unit or more,120,000 cm⁻¹M⁻¹ per repeating unit or more, or even more. In someinstances, the extinction coefficient described herein is an averageextinction coefficient. In certain instances, the repeat unit of themultichromophore may include a single monomer, two co-monomers, or threeor more co-monomers.

In certain embodiments, the multichromophore has a quantum yield of 0.05or more, such as 0.1 or more, 0.15 or more, 0.2 or more, 0.25 or more,0.3 or more, 0.35 or more, 0.4 or more, 0.45 or more, 0.5 or more, 0.6or more, 0.7 or more, or even more. In certain cases, themultichromophore has a quantum yield of 0.1 or more. In certaininstances, the multichromophore has a quantum yield of 0.3 or more.

It is understood that in some cases the multichromophores may includeco-blocks (e.g., n and m co-blocks). The subject multichromophores mayinclude any convenient linear arrangements of n and m co-blocks ofvarious lengths within the structure of the overall polymer. Inaddition, the multichromophores may include any convenient arrangementsof co-monomers within such n and/or m co-blocks. A variety of polymersynthesis methods may be utilized to prepare co-monomers and co-blocksof interest in the preparation of the subject multichromophores. It isunderstood that in some cases, the polymerization methods may produce acomposition including a population of conjugated polymers that includessome variation with respect to the particular length and/or end groups(e.g., terminal groups) present in each CP of the population. Theformulae depicted herein may refer to a single compound or to apopulation or sub-population of polymeric compounds.

In some embodiments, the multichromophore includes a BODIPY-comprisingconjugated segment described by formula (I):

wherein:

B is a BODIPY unit (e.g., as described herein);

M is a π conjugated co-monomer;

each L is an end group; and

n is an integer of 1 to 100,000. In certain instances of formula (I),each L is independently selected from the group consisting of a terminalgroup, a π conjugated segment, a linker and a linked specific bindingmember.

Any convenient π conjugated co-monomers may be utilized in the subjectmultichromophores. As used herein, the term “π conjugated co-monomer”refers to any convenient monomer subunit of a polymer that is capable ofπ conjugation (i.e., delocalization of pi electrons across adjacentunits) to adjacent groups along a polymer backbone. In certainembodiments of formula (I), M is selected from a fused 6-5-6 tricyclicco-monomer, a fluorene co-monomer, a phenylene-vinylene co-monomer, aphenylene-ethynylene co-monomer, a carbazole co-monomer, a C₂-C₁₂ alkyneco-monomer, an arylene-ethynylene co-monomer, a heteroarylene-ethynyleneco-monomer, an arylene co-monomer and a heteroarylene co-monomer. Incertain embodiments of formula (I), M is a phenylene-vinyleneco-monomer. In some instances of formula (I), M is aphenylene-ethynylene co-monomer. In some cases of formula (I), M is acarbazole co-monomer. In certain instances of formula (I), M is a C₂-C₁₂alkyne co-monomer. In certain cases of formula (I), M is a anarylene-ethynylene co-monomer. In some embodiments of formula (I), M isa, a heteroarylene-ethynylene co-monomer. In some instances of formula(I), M is a arylene co-monomer. In some cases of formula (I), M is aheteroarylene co-monomer.

In some embodiments of formula (I), M is a fused 6-5-6 tricyclicco-monomer. A fused 6-5-6 tricyclic co-monomer is a co-monomer includinga tricyclic aromatic group having three fused rings in the configuration6-5-6, i.e. two benzo ring fused to a central 5 membered ring. The5-membered ring may be a carbocycle or a heterocycle and may furtherinclude a sidechain substituent at the ring atom that is not fused to abenzo ring. In certain instances, the fused 6-5-6 tricyclic co-monomeris described by the following structure:

where:

Z is —C(R¹)₂— or —N(R¹)—;

each R is independently H or one or more aryl substituents; and

each R¹ is independently selected from the group consisting of an alkyl,a substituted alkyl, an aralkyl, a substituted aralkyl, a PEG moiety and-L¹-Z¹, where L¹ is a linker and Z¹ is a chemoslective tag (e.g., a tagincluding a chemoslective functional group) or a WSG. As used in any ofthe formulae described herein, * denotes a site for covalent attachmentto the unsaturated backbone of a conjugated polymer or an end group. Insome embodiments, when Z is —N(R¹)—, the fused 6-5-6 tricyclicco-monomer is a carbazole co-monomer. Any convenient carbazoleco-monomers may be utilized in the subject multichromophores. In someembodiments, when Z is —C(R¹)₂—, the fused 6-5-6 tricyclic co-monomer isa fluorene co-monomer. Any convenient fluorene co-monomers may beutilized in the subject multichromophores. In some embodiments offormula (I), M is a carbazole co-monomer. In certain instances of thefused 6-5-6 tricyclic co-monomer, each R¹ is selected from a benzylgroup substituted with one, two or more PEG moieties or an alkyl groupsubstituted with two or more PEG moieties.

In some embodiments of formula (I), M is a fluorene co-monomer. Afluorene co-monomer is an aromatic group having a 9H-fluorene corestructure substituted at the 9 position with any convenient sidechainsubstituent(s). In some cases, the fluorene co-monomer is a9,9-disubstituted fluorene. The fluorene co-monomer may be conjugated toadjacent polymeric backbone groups via any convenient positions of thefluorene core structure, such as any two convenient positions selectedfrom positions 1-8 (see numbering scheme below). In some embodiments,the fluorene core structure is linked to adjacent groups of the polymerbackbone via the 2- and 7-positions (see numbering scheme below). Incertain embodiments, the fluorene co-monomer is described by thefollowing structure:

where each R¹ is independently selected from an alkyl, a substitutedalkyl, an aralkyl, a substituted aralkyl, a PEG moiety and -L¹-Z¹, whereL¹ is a linker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) or a WSG. In certain instances of thefluorene co-monomer, each R¹ is selected from a benzyl group substitutedwith one, two or more PEG moieties or an alkyl group substituted withtwo or more PEG moieties. In some cases, Z¹ includes a functional groupthat finds use in covalently linking the multichromophore to an acceptorchromophore (e.g., as described herein). In certain instances, Z¹includes an amino group for covalently linking to the acceptorchromophore. In certain instances, Z¹ includes a carboxylic acid group,or derivative thereof, for covalently linking to the acceptorchromophore. In certain embodiments, L¹ is a branched linker that linksto two or more Z¹ groups (e.g., WSGs). In certain instances, thefluorene co-monomer is further substituted with a R⁵ and/or R⁶substituent located at one, two or more positions selected frompositions 1, 3, 4, 5, 6 and 8, where R⁵ and R⁶ are independentlyselected from a water solubilizing group (WSG) and an aryl substituent(e.g., as described herein).

In some instances, the fluorene co-monomer is described by thestructure:

where each R² is an alkyl substituted with a water soluble group, or abranched linker connected to two or more water soluble groups (e.g., aPEG-disubstituted benzyl or a PEG substituted alkyl). In certaininstances of the fluorene co-monomer, each R² is a benzyl groupsubstituted with one, two or three PEG moieties (e.g., —O(CH₂CH₂O)_(n)R′where R′ is H or an alkyl and n is 1-20, e.g., 3-16 such as n is 8-16).In certain instances of the fluorene co-monomer, each R² is a benzylgroup substituted with one —O(CH₂CH₂O)_(n)R′ group (e.g., at the 2, 3 or4 position), where R′ is H or an alkyl and n is 1-20, e.g., 3-16 such asn is 8-16. In certain instances of the fluorene co-monomer, each R² is abenzyl group substituted with two —O(CH₂CH₂O)_(n)R′ groups (e.g., at the2,4-, 3,4- or 3,5-positions), where each R′ is independently H or analkyl and each n is independently 1-20, e.g., 3-16 such as n is 8-16. Incertain instances of the fluorene co-monomer, each R² is a benzyl groupsubstituted with three —O(CH₂CH₂O)_(n)R′ groups (e.g., at the 2,4,6-,2,4,5- or 3,4,5-positions), where each R′ is independently H or an alkyland each n is independently 1-20, e.g., 3-16 such as n is 8-16. Incertain instances of the fluorene co-monomer, each R² is a lower alkylgroup substituted with a trivalent branching group each substituted withtwo PEG moieties (e.g., a —CO—NR″₂ or —O(CH₂R″)₂ trivalent branchinggroup where each R″ is independently a PEG moiety (e.g.,—O(CH₂CH₂O)_(n)R′ where R′ is H or an alkyl and n is 1-20, e.g., 3-16such as n is 8-16). In certain embodiments, the fluorene co-monomer isdescribed by the following structure:

where R³ is an alkyl substituted with a water soluble group (e.g., a PEGsubstituted alkyl), and R⁴ is L¹-Z² wherein L¹ is a linker and Z² is achemoselective tag (e.g., for conjugation to an acceptor chromophore) oran acceptor chromophore. Any convenient chemoselective functional groupsmay be included in the subject multichromophores, including, but notlimited to, carboxylic acid, active ester (e.g., NHS or sulfo-NHSester), amino, hydroxyl, thiol, maleimide, iodoacetyl, hydrazido,hydrazino, aldehyde, ketone, azido, alkyne, phosphine, epoxide, and thelike. In some cases, the chemoselective tag is utilized to covalentlylink any convenient moieties (e.g., an acceptor chromophore) to themultichromophore. In certain instances, Z² includes an amino group forcovalently linking to an acceptor chromophore. In certain instances, Z²includes a carboxylic acid group, or derivative thereof, for covalentlylinking to an acceptor chromophore. In certain instances of the fluoreneco-monomer, R³ is a lower alkyl group substituted with a trivalentbranching group each substituted with two PEG moieties (e.g., a —CO—NR″₂or —O(CH₂R″)₂ trivalent branching group where each R″ is a PEG moiety(e.g., —O(CH₂CH₂O)_(n)R′ where R′ is H or an alkyl and n is 1-20, e.g.,3-16 such as n is 8-16).

In some instances, the fluorene co-monomer is described by the followingstructure:

wherein:

R³ is a substituent comprising a water solubilizing group (e.g., asdescribed herein);

R⁴ is L¹-Z² wherein L¹ is a linker and Z² is a chemoselective tag (e.g.,for conjugation to an acceptor chromophore) or an acceptor chromophore;and

R⁵ and R⁶ are independently selected from H, a water solubilizing groupand an aryl substituent (e.g., an alkyl, a substituted alkyl, an alkoxy,a substituted alkoxy, a halogen or a nitro). In certain instances of thefluorene co-monomer, R³ is a lower alkyl group substituted with atrivalent branching group each substituted with two PEG moieties (e.g.,a —CO—NR″₂ or —O(CH₂R″)₂ trivalent branching group where each R″ is aPEG moiety (e.g., —O(CH₂CH₂O)_(n)R′ where R′ is H or an alkyl and n is1-20, e.g., 3-16 such as n is 8-16).

Any convenient end groups may be utilized at the terminals of thesubject multichromphores. End groups of interest include, but are notlimited to a terminal capping group, a π conjugated segment, a linkerand a linked specific binding member. In some embodiments, a terminalcapping group is a monovalent group which is conjugated to the backboneof the multichromophore after polymerization. In certain instances, theterminal capping group is an aryl, a substituted aryl, a heteroaryl, asubstituted heteroaryl, an alkyl or a substituted alkyl. In someembodiments, the terminal capping group is substituted with a linkerand/or a conjugation tag to which any convenient moiety, such as aspecific binding member, may be linked. In certain cases, the end groupis a group derived from a monomer used in the method of polymerization,e.g., a group such as a halogen (e.g., Br), a boronic acid or a boronicester, which is capable of undergoing further conjugation. In someinstances, the end group is a π conjugated segment. As used herein, a πconjugated segment refers to any convenient additional segment of aconjugated polymer to which the multichromophore may be conjugated(i.e., allowing delocalization of pi electron across adjacent units). Incertain embodiments, the end unit is a linker, such as a linkerincluding a functional group suitable for conjugation to a specificbinding moiety. It is understood that linkers and conjugation tagslocated at the terminals of the multichromophore may be selected so asto be orthogonal to any other linkers and chemoselective tags that maybe present at a sidechain of the multichromophore. As used herein, theterms chemoselective tag and conjugation tag, may be usedinterchangeably, and refer to any convenient group that includes afunctional group of interest (e.g., a chemoselective functional group asdescribed herein). In certain embodiments, an amino functional group orderivative thereof is included at an end group (e.g., G¹ and/or G²) anda carboxylic acid functional group or derivative thereof is included atZ¹. In certain embodiments, a carboxylic acid functional group orderivative thereof is included at an end group (e.g., G¹ and/or G²) andan amino functional group or derivative thereof is included at Z¹.

Polymeric Tandem Dyes

As summarized above, the present disclosure provides polymeric tandemdyes that include a light harvesting BODIPY unit-comprisingmultichromophore. Any of the light harvesting BODIPY unit-comprisingmultichromophores described herein may be utilized in the subjectpolymeric tandem dyes. In some embodiments, the polymeric tandem dyesinclude a light harvesting BODIPY unit-comprising multichromophore andan acceptor chromophore covalently linked to the multichromophore inenergy-receiving proximity therewith. In some embodiments, the lightharvesting multichromophore is water soluble.

Polymeric tandem dyes include two covalently linked moieties: a donorlight harvesting multichromophore (e.g., as described herein) and anacceptor chromophore. As used herein, the term “acceptor chromophore”refers to a light-absorbing molecule that is capable of receiving orabsorbing energy transferred from the multichromophore. In some cases,the acceptor chromophore can either emit as light the energy receivedfrom the multichromophore or dissipate the energy as heat. It isunderstood that, unless otherwise stipulated, in the structures andformulae depicted herein, the label “dye” refers to an “acceptorchromophore”. In some instances, the acceptor chromophore is a quencher.As used herein, the term “quencher” refers to an acceptor chromophorethat absorbs energy from the multichromophore and does not emit lightbut rather can dissipate the energy as heat. In certain instances, theacceptor chromophore is a fluorescent dye. In some embodiments, thepolymeric tandem dye may be excited at the absorption maximum wavelengthof the donor multichromophore and may emit light at the emissionwavelength of the acceptor chromophore. In some cases, thelight-harvesting multichromophore can transfer energy to an acceptorchromophore species in energy-receiving proximity. Mechanisms for energytransfer include, for example, resonant energy transfer (e.g., Forster(or fluorescence) resonance energy transfer, FRET), quantum chargeexchange (Dexter energy transfer) and the like. In some instances, theseenergy transfer mechanisms are relatively short range; that is, closeproximity of the light harvesting multichromophore system to theacceptor chromophore provides for efficient energy transfer. In someinstances, under conditions for efficient energy transfer, amplificationof the emission from the acceptor chromophore occurs when the number ofindividual chromophores in the light harvesting multichromophore systemis large; that is, the emission from the signaling chromophore is moreintense when the incident light (the “pump light”) is at a wavelengthwhich is absorbed by the light harvesting multichromophore than when thesignaling chromophore is directly excited by the pump light.

In some cases, by “efficient” energy transfer is meant 5% or more of theenergy harvested is transferred to the acceptor, such as 10% or more,20% or more, 30% or more, 40% or more, 50% or more, or even more. Insome instances, when the acceptor chromophore is a fluorescent dye, theterm efficient energy transfer may refer to a fluorescent quantum yieldof 0.05 or more, such as 0.1 or more, 0.2 or more, 0.3 or more, 0.4 ormore, 0.5 or more, or even greater. By “amplification” is meant that thesignal from the acceptor chromophore is 1.5× or greater when excited bythe light harvesting chromophore as compared to direct excitation withincident light of an equivalent intensity, such as 2.0× or greater, 2.5×or greater, 3× or greater, 4× or greater, 5× or greater, 6× or greater,or greater, 8× or greater, 10× or greater, or even greater. The signalmay be measured using any convenient method. In some cases, the 1.5× orgreater signal refers to an intensity of emitted light. In certaincases, the 1.5× or greater signal refers to an increased signal to noiseratio. In certain embodiments of the polymeric tandem dye, the acceptorchromophore emission is 1.5 fold greater or more when excited by themultichromophore as compared to direct excitation of the acceptorchromophore with incident light.

In some instances, the polymeric tandem dye has an extinctioncoefficient of 5×10⁵ cm⁻¹M⁻¹ or more, such as 6×10⁵ cm⁻¹M⁻¹ or more,7×10⁵ cm⁻¹M⁻¹ or more, 8×10⁵ cm⁻¹M⁻¹ or more, 9×10⁵ cm⁻¹M⁻¹ or more,such as 1×10⁶ cm⁻¹M⁻¹ or more, 1.5×10⁶ cm⁻¹M⁻¹ or more, 2×10⁶ cm⁻¹M⁻¹ ormore, 2.5×10⁶ cm⁻¹M⁻¹ or more, 3×10⁶ cm⁻¹M⁻¹ or more, 4×10⁶ cm⁻¹M⁻¹ ormore, 5×10⁶ cm⁻¹M⁻¹ or more, 6×10⁶ cm⁻¹M⁻¹ or more, 7×10⁶ cm⁻¹M⁻¹ ormore, or 8×10⁶ cm⁻¹M⁻¹ or more. In some embodiments, the polymerictandem dye has a molar extinction coefficient of 5×10⁵ M⁻¹ cm⁻¹ or more.In certain embodiments, the polymeric tandem dye has a molar extinctioncoefficient of 1×10⁶ M⁻¹ cm⁻¹ or more.

In certain embodiments, the polymeric tandem dye has a quantum yield of0.05 or more, such as 0.10 or more, 0.15 or more, 0.20 or more, 0.25 ormore, 0.30 or more 0.35 or more, such as 0.40 or more, 0.45 or more, 0.5or more, or even more. In certain cases, the polymeric tandem dye has aquantum yield of 0.1 or more. In certain instances, the polymeric tandemdye has a quantum yield of 0.3 or more.

Any convenient fluorescent dyes may be utilized in the subject polymerictandem dyes as an acceptor chromophore. The terms “fluorescent dye” and“fluorophore” are used interchangeably herein. In some embodiments, theacceptor chromophore is a cyanine dye, a xanthene dye, a coumarin dye, athiazine dye or an acridine dye. Fluorescent dyes of interest include,but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red,tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP(peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700,7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2,BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY 630/650, BODIPY 650/665, BODIPY R6G, BODIPY TMR, BODIPYTR, conjugates thereof, and combinations thereof. Lanthanide chelates ofinterest include, but are not limited to, europium chelates, terbiumchelates and samarium chelates. In some embodiments, the polymerictandem dye includes a polymeric dye linked to an acceptor fluorophoreselected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647 andAlexa700. In certain embodiments, the polymeric tandem dye includes apolymeric dye linked to an acceptor fluorophore selected from Dyomicsdyes (such as DY 431, DY 485XL, DY 500XL, DY 530, DY 610, DY 633, DY640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800 or DY 831), BiotiumCF 555, Cy 3.5, and diethylamino coumarin.

In certain embodiments of the polymeric tandem dye, the ratio ofco-monomers which lack an acceptor chromophore to co-monomers whichinclude a linked acceptor chromophore is in the range of 40:1 to 3:1,such as in the range of 20:1 to 3:1, 10:1 to 3:1, 9:1 to 3:1, 5:1 to 3:1or 4:1 to 3:1, or in the range of 20:1 to 4:1, 20:1 to 5:1, 20:1 to 9:1or 20:1 to 10:1.

In some instances, the polymeric tandem dye is described by formula(II):

wherein:

B¹ and B² are each independently a BODIPY unit;

each M¹ and each M² are independently a π conjugated co-monomer;

a, b, c, d, e and f are each independently 0, 1 or 2, wherein b+e≧1;

n and m are independently 0 or an integer from 1 to 100,000, whereinn+m≧1;

p is an integer from 1 to 100,000; and

each L² is independently an end group.

In some instances of formula (II), each L² is independently selectedfrom a terminal group, a π conjugated segment, a linker and a linkedspecific binding member. In some embodiments of formula (II), when b is0, a and c are each 1; when e is 0, d and f are each 1; when b is 1,a+c≧1; and when e is 1, d+f≧1. In some embodiments of formula (II), b is1 and e is 0. In some embodiments of formula (II), b is 0 and e is 1. Insome embodiments of formula (II), b is 1 and e is 1. In some embodimentsof formula (II), b is 2 and e is 0. In some embodiments of formula (II),b is 0 and e is 2. In some embodiments of formula (II), b is 2. In someembodiments of formula (II), e is 2.

In some instances of formula (II), at least one of B¹, B², M¹ and M²includes -L¹-C¹, wherein L¹ is an optional linker and C¹ is the acceptorchromophore. In certain instances of formula (II), at least one of B¹and B² includes -L¹-C¹. In some instances of formula (II), at least oneof M¹ and M² includes -L¹-C¹. In some cases of formula (II), M¹ includes-L¹-C¹ and M² does not. In certain cases of formula (II), M² includes-L¹-C¹ and M¹ does not. In certain cases of formula (II), B¹ includes-L¹-C¹ and B² does not. In certain instances of formula (II), B²includes -L¹-C¹ and B¹ does not.

In some embodiments of formula (II), a is 0. In some instances offormula (II), a is 1. In certain embodiments of formula (II), c is 0. Incertain instances of formula (II), c is 1. In some instances of formula(II), a is 1 and c is 0. In certain instances of formula (II), a is 0and c is 1. In certain cases of formula (II), d is 0. In some instancesof formula (II), d is 1. In some embodiments of formula (II), f is 0. Insome instances of formula (II), f is 1. In some instances of formula(II), d is 1 and f is 0. In certain instances of formula (II), d is 0and f is 1.

In some instances, the polymeric tandem dye is described by formula(III):

where B¹, B², M¹, M², n, m, p and each L² is as defined for formula(II), and each L¹ is independently an optional linker and each C¹ isindependently an acceptor chromophore. In some cases of formula (III),each M¹ and each M² are independently a fluorene co-monomer, optionallysubstituted with a water solubilizing group. In some cases of formula(III), each M¹ and each M² are independently a fused 6-5-6 tricyclicco-monomer, such as a fluorene co-monomer or a carbazole co-monomer,optionally substituted with a water solubilizing group. In certainembodiments of formula (III), the ratio of n to m is in the range of20:1 to 3:1, such as in the range of 10:1 to 3:1, 9:1 to 3:1, 5:1 to 3:1or 4:1 to 3:1, or in the range of 20:1 to 4:1, 20:1 to 5:1, 20:1 to 9:1or 20:1 to 10:1.

In some embodiments, the polymeric tandem dye is described by formula(IV):

where B¹, M², n, m, p and each L² is as defined for formula (II), eachL¹ is an optional linker and each C¹ is an acceptor chromophore. Incertain embodiments of formula (IV), each M² is independently a fluoreneco-monomer, optionally substituted with a water solubilizing group. Insome cases of formula (IV), each M² is independently a fused 6-5-6tricyclic co-monomer, such as a fluorene co-monomer or a carbazoleco-monomer, optionally substituted with a water solubilizing group. Incertain instances of formula (IV), the ratio of n to m is in the rangeof 20:1 to 3:1, such as in the range of 10:1 to 3:1, 9:1 to 3:1, 5:1 to3:1 or 4:1 to 3:1, or in the range of 20:1 to 4:1, 20:1 to 5:1, 20:1 to9:1 or 20:1 to 10:1.

In some embodiments, the polymeric tandem dye is described by formula(V):

where B¹, B², M², n, m, p and each L² is as defined for formula (II),and each L² is an optional linker and each C¹ is an acceptorchromophore. In certain embodiments of formula (V), each M² isindependently a fluorene co-monomer, optionally substituted with a watersolubilizing group. In some cases of formula (V), each M² isindependently a fused 6-5-6 tricyclic co-monomer, such as a fluoreneco-monomer or a carbazole co-monomer, optionally substituted with awater solubilizing group. In certain instances of formula (V), the ratioof n to m is in the range of 20:1 to 3:1, such as in the range of 10:1to 3:1, 9:1 to 3:1, 5:1 to 3:1 or 4:1 to 3:1, or in the range of 20:1 to4:1, 20:1 to 5:1, 20:1 to 9:1 or 20:1 to 10:1.

In some embodiments, the polymeric tandem dye is described by formula(VI):

where B¹, M¹, M², n, m, p and each L² is as defined for formula (II)each L¹ is an optional linker and each C¹ is an acceptor chromophore. Incertain embodiments of formula (VI), each M¹ is a carbazole co-monomerand each M² is a fluorene co-monomer, optionally substituted with awater solubilizing group. In some cases of formula (VI), each M¹ andeach M² are independently a fused 6-5-6 tricyclic co-monomer, such as afluorene co-monomer or a carbazole co-monomer, optionally substitutedwith a water solubilizing group. In certain instances of formula (VI),the ratio of n to m is in the range of 20:1 to 3:1, such as in the rangeof 10:1 to 3:1, 9:1 to 3:1, 5:1 to 3:1 or 4:1 to 3:1, or in the range of20:1 to 4:1, 20:1 to 5:1, 20:1 to 9:1 or 20:1 to 10:1.

In some embodiments of formulae (II) to (VI), B¹ and B² are eachindependently described by the following structure:

where R⁶ is an aryl, a heteroaryl or a linker, optionally substitutedwith one or more water solubilizing groups (WSGs). In certain instances,R⁶ is a branched linker that links the BODIPY core structure to two ormore WSGs. In some cases, R⁶ is an aryl or heteroaryl moiety that isfurther substituted with one, two or more WSGs, via optional linkers. Incertain embodiments, R⁶ is a phenyl group substituted with 1, 2, 3 ormore hydrophilic polymer substituents (e.g., a PEG or a modified PEGsubstituent). In certain instances, R⁶ is a phenyl that is substitutedwith one, two or three PEG moieties (e.g., —O(CH₂CH₂O)_(n)R′ where R′ isH or an alkyl and n is 1-20, e.g., 3-16 such as n is 8-16). In certaininstances, R⁶ is a phenyl that is substituted with one —O(CH₂CH₂O)_(n)R′group (e.g., at the 2, 3 or 4 position), where R′ is H or an alkyl and nis 1-20, e.g., 3-16 such as n is 8-16. In certain instances, R⁶ is aphenyl that is substituted with two —O(CH₂CH₂O)_(n)R′ groups (e.g., atthe 2,4-, 3,4- or 3,5-positions), where each R′ is independently H or analkyl and each n is independently 1-20, e.g., 3-16 such as n is 8-16. Incertain instances, R⁶ is a phenyl that is substituted with three—O(CH₂CH₂O)_(n)R′ groups (e.g., at the 2,4,6-, 2,4,5- or3,4,5-positions), where each R′ is independently H or an alkyl and eachn is independently 1-20, e.g., 3-16 such as n is 8-16.

In some instances of formulae (II) to (VI), B¹ and B² are eachindependently described by the following structure:

where each R′ is independently selected from H and an alkyl; and p is 0or an integer from 1-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or12. In certain instances, each R′ is methyl. In some cases, each p is 3.

In some embodiments of formulae (II) to (VI), at least one L² group is-L³-Z where L³ is a linker and Z is a specific binding member (e.g., asdescribed herein). In some embodiments of formulae (II) to (VI), atleast one L² is -L³-Z where L³ is a linker (e.g., as described herein)and Z is a chemoselective tag (e.g., as described herein). In someinstances, Z is selected from carboxylic acid, active ester (e.g.,N-hydroxy succinimidyl ester (NHS) or sulfo-NHS), amino, maleimide,iodoacetyl and thiol. In certain embodiments of formula (II) to (VI), atleast one L² group is described by the following structure:*—Ar-L-Z

where Ar is a π-conjugated aryl or heteroaryl group, L is a linker and Zis a chemoselective tag or a specific binding member. In certainembodiments of formula (II) to (VI), at least one L² group is describedby one of the following structures:

where q is 0 or an integer from 1-12; L is an optional linker; and Z isa chemoselective tag or a specific binding member. In certainembodiments of formula (II) to (VI), at least one L² group is describedby the structure:

where q is 0 or an integer from 1-12; L is an optional linker; and Z isa chemoselective tag or a specific binding member. In certain instances,—NH-L-Z includes an amide linkage to the chemoselective tag or specificbinding member. In certain embodiments, Z is a specific binding memberthat is a biomolecule. In certain instances, Z is an antibody. In someinstances, Z is an antibody fragment or binding derivative thereof. Insome cases, the antibody fragment or binding derivative thereof isselected from a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody anda triabody.

In some embodiments of formulae (II) to (VI), C¹ is selected from acyanine dye, a xanthene dye, a coumarin dye, a thiazine dye and anacridine dye. In certain cases, the linker is selected from an alkyl, asubstituted alkyl, an alkyl-amido, an alkyl-amido-alkyl and a PEGmoiety. In certain embodiments of formulae (II) to (VI), the acceptorchromophore C¹ is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy 3.5 anddiethylamino coumarin. In certain instances of formulae (II) to (VI),the acceptor chromophore is selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7,Alexa488, Alexa 647 and Alexa700.

In some embodiments, the polymeric tandem dye is described by formula(VII):

where:

each q and r is independently an integer from 1-20;

each R′ is independently hydrogen or an alkyl (e.g., methyl);

each R¹ is independently an alkyl, an aryl, a heteroaryl or a linker,optionally substituted with one, two or more water solubilizing groups(WSGs) (e.g., a PEG-disubstituted benzyl or a PEG substituted alkyl);

dye is an acceptor chromophore;

n, m, p and L² are as defined for formula (II); and

L is a linker and Z is a chemoselective tag or a specific bindingmember. In certain cases for formula (VII), L is —O—(CH₂)_(n)—NH—, wheren is 2-12, such as n is 4. In certain instances, each R¹ is described bythe structure:

where each q is independently an integer from 2-20, and each R′ isindependently hydrogen, an alkyl or a substituted alkyl. In certaincases, each R′ is methyl. In some cases, each q is 2. In certain cases,each q is 3. In certain cases, each q is 4. In certain cases, each q is5. In certain cases, each q is 6. In certain cases, each q is 7. Incertain cases, each q is 8. In certain cases, each q is 9. In certaincases, each q is 10. In certain cases, each q is 11. In certaininstances, L² is a terminal group.

In some embodiments, the polymeric tandem dye is described by formula(VIII):

where:

each q and r is independently an integer from 1-20;

each R′ is independently hydrogen, an alkyl or a substituted alkyl;

n, m, p and L² are as defined for formula (II);

each R¹ is independently an alkyl, an aryl, a heteroaryl or a linker,optionally substituted with one, two or more water solubilizing groups(WSGs) (e.g., a PEG-disubstituted benzyl or a PEG substituted alkyl);

dye is an acceptor chromophore;

L is a linker and Z is a chemoselective tag or a specific bindingmember. In certain cases, each R′ is methyl. In certain cases of formula(VIII), L-Z is —O—(CH₂)_(n)—NH₂, where n is 2-12 (e.g., n is 2, 3 or 4).In certain cases of formula (VIII), L is —O—(CH₂)_(n)—NH—, where n is2-12, such as n is 4, and Z is a specific binding member (e.g., abiomolecule). In certain cases of formula (VIII), L-Z is —(CH₂)_(n)—NH₂,where n is 2-12 (e.g., n is 2, 3 or 4). In certain cases of formula(VIII), L is —(CH₂)_(n)—NH— and Z is a specific binding member (e.g., abiomolecule), where n is 2-12 (e.g., n is 2, 3 or 4). It is understoodthat the Dye group of formula (VIII) which is linked to the fluoreneco-monomer via a —CONH-Dye linkage may alternatively be linked via witha —NHCO-Dye connection. In such alternate depiction of formula (VIII), Lcan be —(CH₂)_(n)—CO—, where n is 2-12 (e.g., n is 2, 3 or 4). Incertain instances, each R¹ is described by the structure:

where each q is independently an integer from 2-20, and each R′ isindependently hydrogen, an alkyl or a substituted alkyl. In certaincases, each R′ is methyl. In some cases, each q is 2. In certain cases,each q is 3. In certain cases, each q is 4. In certain cases, each q is5. In certain cases, each q is 6. In certain cases, each q is 7. Incertain cases, each q is 8. In certain cases, each q is 9. In certaincases, each q is 10. In certain cases, each q is 11. In certaininstances, L² is a terminal group.

In some embodiments, the polymeric tandem dye is described by formula(IX):

where:

each q is independently an integer from 1-20;

each R′ is independently hydrogen, an alkyl or a substituted alkyl;

each R¹ is independently an alkyl, an aryl, a heteroaryl or a linker,optionally substituted with one, two or more water solubilizing groups(WSGs) (e.g., a PEG-disubstituted benzyl or a PEG substituted alkyl);

dye is an acceptor chromophore;

n, m, p and L² are as defined in formula (II);

L is a linker and Z is a chemoselective tag or a specific bindingmember. In certain cases, each R′ is methyl. In certain cases forformula (IX), L is —O—(CH₂)_(n)—NH—, where n is 2-12, such as n is 4. Incertain instances, each R¹ is described by the structure:

where each q is independently an integer from 2-20, and each R′ isindependently hydrogen, an alkyl or a substituted alkyl. In certaincases, each R′ is methyl. In some cases, each q is 2. In certain cases,each q is 3. In certain cases, each q is 4. In certain cases, each q is5. In certain cases, each q is 6. In certain cases, each q is 7. Incertain cases, each q is 8. In certain cases, each q is 9. In certaincases, each q is 10. In certain cases, each q is 11. In certaininstances, L² is a terminal group.

In some embodiments, the polymeric tandem dye is described by formula(X):

where:

each PEGn is independently a PEG or a modified PEG of from 1-20 units;

each R¹ is independently an alkyl, an aryl, a heteroaryl or a linker,optionally substituted with one, two or more water solubilizing groups(WSGs) (e.g., a PEG-disubstituted benzyl or a PEG substituted alkyl);

dye is an acceptor chromophore;

n, m, p and L² are as defined in formula (II);

each L is a linker and Z is a chemoselective tag or a specific bindingmember. In certain cases of formula (X), L is —O—(CH₂)_(n)—NH—, where nis 2-12, such as n is 4. In certain instances of formula (X), R¹ is analkyl. In certain embodiments of formula (X), the ratio of n to m is inthe range of 20:1 to 3:1, such as 15:1 to 4:1, 10:1 to 4:1, or 9:1 to5:1.

In some embodiments, the polymeric tandem dye is described by formula(XI):

where:

each q is independently an integer from 1-20;

each R′ is independently hydrogen, an alkyl or a substituted alkyl;

dye is an acceptor chromophore;

n, m, p and L² are as defined in formula (II);

each L is a linker and Z is a chemoselective tag or a specific bindingmember. In certain cases of formula (XI), L is —O—(CH₂)_(n)—NH—, where nis 2-12, such as n is 4. In certain cases of formula (XI), L is—(CH₂)_(n)—CONH—, where n is 1-12, such as n is 1.

In some embodiments, the polymeric tandem dye is described by formula(XII):

where:

each q is independently an integer from 1-20;

each R′ is independently hydrogen, an alkyl or a substituted alkyl;

dye is an acceptor chromophore;

n, m, p and L² are as defined in formula (II);

each L is a linker and Z is a chemoselective tag or a specific bindingmember. In certain cases of formula (XII), L is —O—(CH₂)_(n)—NH—, wheren is 2-12, such as n is 4. In certain cases of formula (XII), L is—(CH₂)_(n)—CONH—, where n is 1-12, such as n is 1.

In certain instances of the fluorene co-monomer of any one of formulae(II)-(XII), each R¹ or R² sidechain group is a benzyl group substitutedwith one, two or three PEG moieties (e.g., —O(CH₂CH₂O)_(n)R′ where R′ isH or an alkyl and n is 1-20, e.g., 3-16 such as n is 8-16). In certaininstances of the fluorene co-monomer of any one of formulae (II)-(XII),each R¹ or R² sidechain group is a benzyl group substituted with one—O(CH₂CH₂O)_(n)R′ group (e.g., at the 2, 3 or 4 position), where R′ is Hor an alkyl and n is 1-20, e.g., 3-16 such as n is 8-16. In certaininstances of fluorene co-monomer of any one of formulae (II)-(XII), eachR¹ or R² sidechain group is a benzyl group substituted with two—O(CH₂CH₂O)_(n)R′ groups (e.g., at the 2,4-, 3,4- or 3,5-positions),where each R′ is independently H or an alkyl and each n is independently1-20, e.g., 3-16 such as n is 8-16. In certain instances of the fluoreneco-monomer of any one of formulae (II)-(XII), each R¹ or R² sidechaingroup is a benzyl group substituted with three —O(CH₂CH₂O)_(n)R′ groups(e.g., at the 2,4,6-, 2,4,5- or 3,4,5-positions), where each R′ isindependently H or an alkyl and each n is independently 1-20, e.g., 3-16such as n is 8-16. In certain instances of the fluorene co-monomer ofany one of formulae (II)-(XII), each R¹ or R² sidechain group is a loweralkyl group substituted with a trivalent branching group eachsubstituted with two PEG moieties (e.g., a —CO—NR″₂ or —O(CH₂R″)₂trivalent branching group where each R″ is independently a PEG moiety(e.g., —O(CH₂CH₂O)_(n)R′ where R′ is H or an alkyl and n is 1-20, e.g.,3-16 such as n is 8-16).

It is understood that the polymeric tandem dye of any one of formulae(I) to (XII) can alternatively be represented by a formula whichindicates what the mol % values for each co-monomer is in the polymer.For example, in some cases, any one of Formulae (II) to (XII) can berepresented by one of the following formula:L²-(B¹)_(x)(M¹)_(y)(M²-L¹-C¹)_(z)-L²L²-(B¹)_(x)(M²)_(y)(B²-L¹-C¹)_(z)-L²L²-(B¹)_(x)(M²)_(y)(M²-L¹-C¹)_(z)-L²L²-(B¹)_(x)(M²)_(y)(M¹-L¹-C¹)_(z)-L²

where x, y and z are the mol % values of the co-monomers in theconjugated polymer. In some instances of the formulae, x is 1 mol % ormore, such as 2 mol % or more, 3 mol % or more, 4 mol % or more, 5 mol %or more, 10 mol % or more, 15 mol % or more, 20 mol % or more, 25 mol %or more, 30 mol % or more, 35 mol % or more, 40 mol % or more, 45 mol %or more, 50 mol % or more, or even more. In certain instances of theformulae, x ranges from 1 mol % to 50 mol %, such as from 5 mol % to 25mol % or from 10 mol % to 25 mol %; or such as from 5 mol % to 25 mol %or from 10 mol % to 25 mol %; or such as from 1 mol % to 25 mol %, from1 mol % to 10 mol %, or from 1 mol % to 5 mol %. In some instances ofthe formulae, z is 10 mol % or more, such as 15 mol % or more, 20 mol %or more, 25 mol % or more, 30 mol % or more, 35 mol % or more, 40 mol %or more, 45 mol % or more, 50 mol % or more, or even more. In someinstances of the formulae, z is 25 mol % or less, such as 20 mol % orless, 15 mol % or less, 10 mol % or less, 8 mol % or less, 6 mol % orless, 5 mol % or less, 2 mol % or less, 1 mol % or less, or even less.In some instances of the formulae, y is 1 mol % or more, such as 5 mol %or more, 10 mol % or more, 15 mol % or more, 20 mol % or more, or 25 mol% or more. In some instances of the formulae, y is 25 mol % or less,such as 20 mol % or less, 15 mol % or less, 10 mol % or less, 8 mol % orless, 6 mol % or less, 5 mol % or less, 2 mol % or less, 1 mol % orless, or even less.

It is understood that for any of the structures and formula depictedherein that in some cases of the subject polymeric tandem dyes the endor terminal groups depicted may be located at the opposite ends to thoseshown, e.g., the end groups may be switched. In some embodiments of themultichromophores described herein (e.g., formulae (I)-(XII), at leastone end group (e.g., L, L², G¹, G², L-Z) is selected from one of thefollowing structures 1-33:

*=site for covalent attachment to unsaturated backbone;

-   wherein R′ is independently H, halogen, C₁-C₁₂ alkyl,    (C₁-C₁₂alkyl)NH₂, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂cycloalkyl,    C₁-C₁₂ haloalkyl, C₂-C₁₈(hetero)aryl, C₂-C₁₈(hetero)arylamino,    —[CH₂—CH₂]_(r)—Z¹, or (C₁-C₁₂)alkoxy-X¹; and wherein Z¹ is —OH or    —COOH; X¹ is —NH₂, —NHCOOH, —NHCOOC(CH₃)₃,    —NHCO(C3-C12)cycloalkyl(C1-C4)alkyl-N-maleimide; or    —NHCO[CH₂—CH₂—O]_(s′)(CH₂)_(s′)NH₂; r′ is an integer from 1 to 20;    and each s′ is independently an integer from 1 to 20,    (CH₂)₃(OCH₂CH₂)_(x″)OCH₃ where x″ is independently an integer from 0    to 50, or a benzyl optionally substituted with one or more halogen,    hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_(y″)CH₃ where each y″ is    independently an integer from 0 to 50 and R′ is different from R;    wherein k is 2, 4, 8, 12 or 24; wherein R¹⁵ is selected from groups    I-u having the structure:

*=site for covalent attachment to backbone.

In some embodiments of the multichromophores described herein (e.g.,formulae (I)-(XII), at least one end group (e.g., L, L², G¹, G², L-Z) isselected from one of the following structures:

where r is 0 or an integer from 1-50 (e.g., 1-20); k is 0 or an integerfrom 1-50 (e.g., 1-20); R¹ is as defined for any of the fluoreneco-monomers described herein; and R¹⁶ is selected from H, OH, NH₂,—NH(CH₂)r-NH₂, and —NH(CH₂)_(r)COOH.Labelled Specific Binding Members

Aspects of the present disclosure include labelled specific bindingmembers. A labelled specific binding member is a conjugate of a subjectBODIPY unit-comprising multichromophore (e.g., as described herein) anda specific binding member. The multichromophore may be a polymeric dye.The multichromophore may be polymeric tandem dye. The specific bindingmember and the multichromophore may be conjugated (e.g., covalentlylinked) to each other via any convenient locations of themultichromophore, via an optional linker.

As used herein, the term “specific binding member” refers to one memberof a pair of molecules which have binding specificity for one another.One member of the pair of molecules may have an area on its surface, ora cavity, which specifically binds to an area on the surface of, or acavity in, the other member of the pair of molecules. Thus the membersof the pair have the property of binding specifically to each other toproduce a binding complex. In some embodiments, the affinity betweenspecific binding members in a binding complex is characterized by aK_(d) (dissociation constant) of 10⁻⁶ M or less, such as 10⁻⁷ M or less,including 10⁻⁸ M or less, e.g., 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ Mor less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M or less, including10⁻¹⁵ M or less. In some embodiments, the specific binding membersspecifically bind with high avidity. By high avidity is meant that thebinding member specifically binds with an apparent affinitycharacterized by an apparent K_(d) of 10×10⁻⁹ M or less, such as 1×10⁻⁹M or less, 3×10⁻¹⁰ M or less, 1×10⁻¹⁰ M or less, 3×10⁻¹¹ M or less,1×10⁻¹¹ M or less, 3×10⁻¹² M or less or 1×10⁻¹² M or less.

As used herein, the term “proteinaceous” refers to a moiety (e.g., aspecific binding member) that is composed of amino acid residues. Aproteinaceous moiety may be a polypeptide. In some embodiments, thespecific binding member is proteinaceous. In certain cases, theproteinaceous specific binding member is an antibody. In certainembodiments, the proteinaceous specific binding member is an antibodyfragment, e.g., a binding fragment of an antibody that specific binds toa polymeric dye. As used herein, the terms “antibody” and “antibodymolecule” are used interchangeably and refer to a protein consisting ofone or more polypeptides substantially encoded by all or part of therecognized immunoglobulin genes. The recognized immunoglobulin genes,for example in humans, include the kappa (k), lambda (l), and heavychain genetic loci, which together comprise the myriad variable regiongenes, and the constant region genes mu (u), delta (d), gamma (g), sigma(e), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypesrespectively. An immunoglobulin light or heavy chain variable regionconsists of a “framework” region (FR) interrupted by three hypervariableregions, also called “complementarity determining regions” or “CDRs”.The extent of the framework region and CDRs have been precisely defined(see, “Sequences of Proteins of Immunological Interest,” E. Kabat etal., U.S. Department of Health and Human Services, (1991)). Thenumbering of all antibody amino acid sequences discussed herein conformsto the Kabat system. The sequences of the framework regions of differentlight or heavy chains are relatively conserved within a species. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs. The CDRs are primarily responsible for binding to an epitopeof an antigen.

The term antibody is meant to include full length antibodies and mayrefer to a natural antibody from any organism, an engineered antibody,or an antibody generated recombinantly for experimental, therapeutic, orother purposes as further defined below. Antibody fragments of interestinclude, but are not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, or otherantigen-binding subsequences of antibodies, either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies. Antibodies may be monoclonal or polyclonaland may have other specific activities on cells (e.g., antagonists,agonists, neutralizing, inhibitory, or stimulatory antibodies). It isunderstood that the antibodies may have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other antibody functions.

In certain embodiments, the specific binding member is an antibody. Incertain embodiments, the specific binding member is a Fab fragment, aF(ab′)₂ fragment, a scFv, a diabody or a triabody. In some cases, thespecific binding member is a murine antibody or binding fragmentthereof. In certain instances, the specific binding member is arecombinant antibody or binding fragment thereof.

In some embodiments, the labelled specific binding member includes: apolymeric tandem dye including: a light harvesting BODIPYunit-comprising multichromophore; an acceptor chromophore covalentlylinked to the multichromophore in energy-receiving proximity therewith;and a specific binding member covalently linked to the multichromophore.In certain instances of the labelled specific binding member, the lightharvesting multichromophore is water soluble. In some instances of thelabelled specific binding member, the dye has narrow band spectralfeatures. In some instances of the labelled specific binding member, thedye has low energy absorption bands having a bandwidth of 100 nm orless, such as 50 nm or less. In certain instances of the labelledspecific binding member, the multichromophore has a molar extinctioncoefficient of 5×10⁵ M⁻¹ cm⁻¹ or more (e.g., as described herein). Incertain cases of the labelled specific binding member, themultichromophore has a quantum yield of 0.05 or more (e.g., as describedherein). In some embodiments, the labelled specific binding memberfurther includes an acceptor chromophore covalently linked to themultichromophore in energy-receiving proximity therewith, e.g., themultichromophore is a polymeric tandem dye. In some embodiments of thelabelled specific binding member, the dye has a ratio of acceptorchromophores to multichromophore repeat units in the range of 1:40 to1:4, such as a ratio of in the range of 1:20 to 1:4, 1:10 to 1:4, 1:9 to1:4, 1:8 to 1:4, 1:7 to 1:4, 1:6 to 1:4, or 1:5 to 1:4, or such as aratio in the range of 1:40 to 1:5, 1:40 to 1:6, 1:40 to 1:7, 1:40 to1:8, 1:40 to 1:9, 1:40 to 1:10, or 1:40 to 1:20.

In certain cases, the acceptor chromophore is a fluorophore. In someembodiments of the labelled specific binding member, the acceptorchromophore emission is 1.5-fold greater or more (such as 2.0-foldgreater or more, 2.5-fold greater or more, 3-fold greater or more,4-fold greater or more, 5-fold greater or more, 6-fold greater or more,7-fold greater or more, 8-fold greater or more, 9-fold greater or more,10-fold greater or more, or even more) when excited by themultichromophore as compared to direct excitation of the acceptorchromophore with incident light.

In some instances of the labelled specific binding member, themultichromophore includes a BODIPY-comprising conjugated segmentdescribed by formula (I):

where B is a BODIPY unit; M is a π conjugated co-monomer; each L isindependently selected from a terminal group, a π conjugated segment, alinker and a linked specific binding member; and n is an integer of 1 to100,000. In certain embodiments of formula (I), the BODIPY unit isdescribed by the structure:

where: R¹, R², R³ and R⁴ are each independently selected from H, analkyl and a substituted alkyl; R⁵ is selected from an alkyl, asubstituted alkyl, an aryl, a substituted aryl, a heteroaryl and asubstituted heteroaryl, wherein R⁵ is optionally substituted with awater solubilizing group; and each R is selected from F, OH, H, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, alkynyl and substituted alkynyl.In certain embodiments of formula (I), M is selected from a fluoreneco-monomer, a phenylene-vinylene co-monomer, a phenylene-ethynyleneco-monomer, a carbazole co-monomer, a C₂-C₁₂ alkyne co-monomer, anarylene-ethynylene co-monomer, a heteroarylene-ethynylene co-monomer, anarylene co-monomer and a heteroarylene co-monomer.

In some embodiments of the labelled specific binding member, themultichromophore is described by the formula (II):

where: B¹ and B² are each independently a BODIPY unit; each M¹ and eachM² are independently a π conjugated co-monomer; a, b, c, d, e and f areeach independently 0, 1 or 2, wherein b+e≧1; n and m are independently 0to 100,000, wherein n+m≧1; and one L² group is a terminal group (G¹) andthe other L² group is a linked specific binding member (e.g., L-Z). Incertain embodiments of formula (II), at least one of B¹, B², M¹ and M²includes -L¹-C¹, wherein L¹ is an optional linker and C¹ is the acceptorchromophore.

In certain embodiments of formula (II), the linked specific bindingmember is an antibody. In some instances of formula (II), the linkedspecific binding member is an antibody fragment or binding derivativethereof. In some cases of formula (II), the linked specific bindingmember is an antibody fragment or binding derivative thereof that isselected from a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody anda triabody. In some instances of formula (II), the acceptor chromophoreis selected from a cyanine dye, a xanthene dye, a coumarin dye, athiazine dye and an acridine dye. In certain instances of formula (II),the acceptor chromophore is selected from DY 431, DY 485XL, DY 500XL, DY610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy3.5 and diethylamino coumarin. In some instances of formula (II), theacceptor chromophore is selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7,Alexa488, Alexa 647 and Alexa700.

In some embodiments, the labelled specific binding member is a polymerictandem dye of formula (III), where one L² group is a linked specificbinding member. In some embodiments, the labelled specific bindingmember is a polymeric tandem dye of formula (IV), where one L² group isa linked specific binding member. In some embodiments, the labelledspecific binding member is a polymeric tandem dye of formula (V), whereone L² group is a linked specific binding member. In some embodiments,the labelled specific binding member is a polymeric tandem dye offormula (VI), where one L² group is a linked specific binding member.

In some embodiments, the labelled specific binding member is a polymerictandem dye of formula (VII), where Z is a specific binding member. Insome embodiments, the labelled specific binding member is a polymerictandem dye of formula (VIII), where Z is a specific binding member. Insome embodiments, the labelled specific binding member is a polymerictandem dye of formula (IX), where Z is a specific binding member. Insome embodiments, the labelled specific binding member is a polymerictandem dye of formula (X), where Z is a specific binding member. In someembodiments, the labelled specific binding member is a polymeric tandemdye of formula (XI), where Z is a specific binding member. In someembodiments, the labelled specific binding member is a polymeric tandemdye of formula (XII), where Z is a specific binding member.

In certain embodiments, the labelled specific binding member isdescribed by the following structure:

In certain embodiments, the labelled specific binding member isdescribed by the following structure:

In certain embodiments, the labelled specific binding member isdescribed by the following structure:

In certain embodiments, the labelled specific binding member isdescribed by the following structure:

In certain embodiments, the labelled specific binding member isdescribed by the following structure:

In certain embodiments, the labelled specific binding member isdescribed by the following structure:

Also provided are polymeric tandem dye precursors of any one of thestructures shown above which include a terminal amino functional groupsuitable for conjugation to the “Biomolecule”. The structures of suchpolymeric tandem dye precursors may be represented by replacing the“Biomolecule” group depicted in the structures above with an “H”. Insome instances of the structures depicted above, the linked “dye” is alinked fluorescent dye.

Methods

As summarized above, aspects of the invention include methods ofevaluating a sample for the presence of a target analyte. In someembodiments, the method includes: (a) contacting the sample with apolymeric dye conjugate that specifically binds the target analyte toproduce a labelling composition contacted sample; and (b) assaying thelabelling composition contacted sample for the presence of a polymericdye conjugate-target analyte binding complex to evaluate whether thetarget analyte is present in the sample. In certain embodiments of themethod, the polymeric dye conjugate includes: (i) a light harvestingBODIPY unit-comprising multichromophore (e.g., as described herein);(ii) an acceptor chromophore covalently linked to the multichromophorein energy-receiving proximity therewith; and (iii) a specific bindingmember (e.g., as described herein).

Any convenient method may be used to contact the sample with a polymericdye conjugate that specifically binds to the target analyte to producethe labelling composition contacted sample. As used herein, the terms“polymeric dye conjugate” and “labelled specific binding member” areused interchangeably. In some instances, the sample is contacted withthe polymeric dye conjugate under conditions in which the specificbinding member specifically binds to the target analyte, if present. Forspecific binding of the specific binding member of the conjugate withthe target analyte, an appropriate solution may be used that maintainsthe biological activity of the components of the sample and the specificbinding member. The solution may be a balanced salt solution, e.g.,normal saline, PBS, Hank's balanced salt solution, etc., convenientlysupplemented with fetal calf serum, human platelet lysate or otherfactors, in conjunction with an acceptable buffer at low concentration,such as from 5-25 mM. Convenient buffers include HEPES, phosphatebuffers, lactate buffers, etc. Various media are commercially availableand may be used according to the nature of the target analyte, includingdMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., in some casessupplemented with fetal calf serum or human platelet lysate. The finalcomponents of the solution may be selected depending on the componentsof the sample which are included.

The temperature at which specific binding of the specific binding memberof the conjugate to the target analyte takes place may vary, and in someinstances may range from 5° C. to 50° C., such as from 10° C. to 40° C.,15° C. to 40° C., 20° C. to 40° C., e.g., 20° C., 25° C., 30° C., 35° C.or 37° C. (e.g., as described above). In some instances, the temperatureat which specific binding takes place is selected to be compatible withthe biological activity of the specific binding member and/or the targetanalyte. In certain instances, the temperature is 25° C., 30° C., 35° C.or 37° C. In certain cases, the specific binding member is an antibodyor fragment thereof and the temperature at which specific binding takesplace is room temperature (e.g., 25° C.), 30° C., 35° C. or 37° C. Anyconvenient incubation time for specific binding may be selected to allowfor the formation of a desirable amount of binding complex, and in someinstances, may be 1 minute (min) or more, such as 2 min or more, 10 minor more, 30 min or more, 1 hour or more, 2 hours or more, or even 6hours or more.

Any convenient specific binding members may be utilized in the polymericdye conjugate. Specific binding members of interest include, but are notlimited to, those agents that specifically bind cell surface proteins ofa variety of cell types, including but not limited to, stem cells, e.g.,pluripotent stem cells, hematopoietic stem cells, T cells, T regulatorcells, dendritic cells, B Cells, e.g., memory B cells, antigen specificB cells, granulocytes, leukemia cells, lymphoma cells, virus cells(e.g., HIV cells) NK cells, macrophages, monocytes, fibroblasts,epithelial cells, endothelial cells, and erythroid cells. Target cellsof interest include cells that have a convenient cell surface marker orantigen that may be captured by a convenient specific binding memberconjugate. In some embodiments, the target cell is selected from HIVcontaining cell, a Treg cell, an antigen-specific T-cell populations,tumor cells or hematopoetic progenitor cells (CD34+) from whole blood,bone marrow or cord blood. Any convenient cell surface proteins or cellmarkers may be targeted for specific binding to polymeric dye conjugatesin the subject methods. In some embodiments, the target cell includes acell surface marker selected from a cell receptor and a cell surfaceantigen. In some cases, the target cell may include a cell surfaceantigen such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25,CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34,CD1c, CD23, CD304, CD235a, T cell receptor alpha/beta, T cell receptorgamma/delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5(N-Terminal), SSEA-3, TRA-1-60 Antigen, Disialoganglioside GD2 and CD71.

Any convenient targets may be selected for evaluation utilizing thesubject methods. Targets of interest include, but are not limited to, anucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule, aprotein, such as a fusion protein, a modified protein, such as aphosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylatedprotein, or an antibody, a peptide, an aggregated biomolecule, a cell, asmall molecule, a vitamin and a drug molecule. As used herein, the term“a target protein” refers to all members of the target family, andfragments thereof. The target protein may be any protein of interest,such as a therapeutic or diagnostic target, including but not limitedto: hormones, growth factors, receptors, enzymes, cytokines,osteoinductive factors, colony stimulating factors and immunoglobulins.The term “target protein” is intended to include recombinant andsynthetic molecules, which can be prepared using any convenientrecombinant expression methods or using any convenient syntheticmethods, or purchased commercially. In some embodiments, the polymericdye conjugates include an antibody or antibody fragment. Any convenienttarget analyte that specifically binds an antibody or antibody fragmentof interest may be targeted in the subject methods.

In some embodiments, the target analyte is associated with a cell. Incertain instances, the target analyte is a cell surface marker of thecell. In certain cases, the cell surface marker is selected from a cellreceptor and a cell surface antigen. In some instances, the targetanalyte is an intracellular target, and the method further includeslysing the cell. In certain instances, the method further includesextracting proteins from the cell. Any convenient methods and agents maybe utilized in lysing the cell. Methods and agents of interest includethose cell lysing and protein extraction methods and agents described atwww.piercenet.com/method/traditional-methods-cell-lysis.

In some embodiments, the sample may include a heterogeneous cellpopulation from which target cells are isolated. In some instances, thesample includes peripheral whole blood, peripheral whole blood in whicherythrocytes have been lysed prior to cell isolation, cord blood, bonemarrow, density gradient-purified peripheral blood mononuclear cells orhomogenized tissue. In some cases, the sample includes hematopoeticprogenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cordblood. In certain embodiments, the sample includes tumor cells inperipheral blood. In certain instances, the sample is a sample including(or suspected of including) viral cells (e.g., HIV).

The subject labelled specific binding members find use in the subjectmethods, e.g., for labeling a target cell, particle, target or analytewith a polymeric dye or polymeric tandem dye. For example, labelledspecific binding members find use in labeling cells to be processed(e.g., detected, analyzed, and/or sorted) in a flow cytometer. Thelabelled specific binding members may include antibodies thatspecifically bind to, e.g., cell surface proteins of a variety of celltypes (e.g., as described herein). The labelled specific binding membersmay be used to investigate a variety of biological (e.g., cellular)properties or processes such as cell cycle, cell proliferation, celldifferentiation, DNA repair, T cell signaling, apoptosis, cell surfaceprotein expression and/or presentation, and so forth. labelled specificbinding members may be used in any application that includes (or mayinclude) antibody-mediated labeling of a cell, particle or analyte.

In some embodiments, the polymeric dye conjugate includes a polymerictandem dye (e.g., as described herein). As such, in some embodiments,the polymeric dye conjugate further includes an acceptor chromophorecovalently linked to the multichromophore in energy-receiving proximitytherewith. In certain embodiments, the conjugate is described by formula(II):

where B¹ and B² are each independently a BODIPY unit; each M¹ and eachM² are independently a π conjugated co-monomer; a, b, c, d, e and f areeach independently 0, 1 or 2, wherein b+e≧1; n and m are independently 0to 100,000, wherein n+m≧1; and one L² group is a terminal group (G¹) andthe other L² group is a linked specific binding member. In certainembodiments, at least one of B¹, B², M¹ and M² includes -L¹-C¹, whereinL¹ is an optional linker and C¹ is the acceptor chromophore.

Once the sample has been contacted with the polymeric dye conjugate, anyconvenient methods may be utilized in assaying the labelling compositioncontacted sample that is produced for the presence of a polymeric dyeconjugate-target analyte binding complex. The polymeric dyeconjugate-target analyte binding complex is the binding complex that isproduced upon specific binding of the specific binding member of theconjugate to the target analyte, if present. Assaying the labellingcomposition contacted sample may include detecting a fluorescent signalfrom the binding complex, if present. In some cases, the assayingincludes a separating step where the target analyte, if present, isseparated from the sample. A variety of methods may be utilized toseparate a target analyte from a sample, e.g., via immobilization on asupport. Assay methods of interest include, but are not limited to, anyconvenient methods and assay formats where pairs of specific bindingmembers such as avidin-biotin or hapten-anti-hapten antibodies find use,are of interest. Methods and assay formats of interest that may beadapted for use with the subject compositions include, but are notlimited to, flow cytometry methods, in-situ hybridization methods,enzyme-linked immunosorbent assays (ELISAs), western blot analysis,magnetic cell separation assays and fluorochrome purificationchromatography.

In certain embodiments, the method further includes contacting thesample with a second specific binding member that specifically binds thetarget analyte. In certain instances, the second specific binding memberis support bound. Any convenient supports may be utilized to immobilizea component of the subject methods (e.g., a second specific bindingmember). In certain instances, the support is a particle, such as amagnetic particle. In some instances, the second specific binding memberand the polymeric dye conjugate produce a sandwich complex that may beisolated and detected, if present, using any convenient methods. In someembodiments, the method further includes flow cytometrically analyzingthe polymeric dye conjugate-target analyte binding complex, i.e., afluorescently labelled target analyte. Assaying for the presence of apolymeric dye conjugate-target analyte binding complex may provide assayresults (e.g., qualitative or quantitative assay data) which can be usedto evaluate whether the target analyte is present in the sample.

Any convenient supports may be utilized in the subject methods. Supportsof interest include, but are not limited to: solid substrates, where thesubstrate can have a variety of configurations, e.g., a sheet, bead, orother structure, such as a plate with wells; beads, polymers, particle,a fibrous mesh, hydrogels, porous matrix, a pin, a microarray surface, achromatography support, and the like. In some instances, the support isselected from a particle, a planar solid substrate, a fibrous mesh, ahydrogel, a porous matrix, a pin, a microarray surface and achromatography support. The support may be incorporated into a systemthat it provides for cell isolation assisted by any convenient methods,such as a manually-operated syringe, a centrifuge or an automated liquidhandling system. In some cases, the support finds use in an automatedliquid handling system for the high throughput isolation of cells, suchas a flow cytometer.

In some embodiments of the method, the separating step includes applyingan external magnetic field to immobilize a magnetic particle. Anyconvenient magnet may be used as a source of the external magnetic field(e.g., magnetic field gradient). In some cases, the external magneticfield is generated by a magnetic source, e.g. by a permanent magnet orelectromagnet. In some cases, immobilizing the magnetic particles meansthe magnetic particles accumulate near the surface closest to themagnetic field gradient source, i.e. the magnet.

The separating may further include one or more optional washing steps toremove unbound material of the sample from the support. Any convenientwashing methods may be used, e.g., washing the immobilized support witha biocompatible buffer which preserves the specific binding interactionof the polymeric dye and the specific binding member. Separation andoptional washing of unbound material of the sample from the supportprovides for an enriched population of target cells where undesiredcells and material may be removed.

In certain embodiments, the method further includes detecting thelabelled target. Detecting the labelled target may include exciting themultichromophore with one or more lasers and subsequently detectingfluorescence emission from the polymeric dye using one or more opticaldetectors.

Also provided are methods of labelled a target molecule. The subjectpolymeric dyes, including tandem dyes, find use in a variety of methodsof labelling, separation, detection and/or analysis. In someembodiments, the method includes: contacting the target molecule with apolymeric tandem dye to produce a labelled target molecule, wherein thepolymeric tandem dye includes: a light harvesting BODIPY unit-comprisingmultichromophore (e.g., as described herein); and a conjugation tag. Incertain instances, the polymeric dye is itself fluorescent. In someembodiments, the polymeric dye is a polymeric tandem dye. As such, insome cases, the polymeric dye further includes an acceptor chromophorecovalently linked to the multichromophore in energy-receiving proximitytherewith. As used herein, the term “labelled target molecule” refers toa target molecule that is covalently linked to a subjectmultichromophore.

In some embodiments, the polymeric dye is described by formula (II):

where: B¹ and B² are each independently a BODIPY unit; each M¹ and eachM² are independently a π conjugated co-monomer; a, b, c, d, e and f areeach independently 0, 1 or 2, wherein b+e≧1; n and m are independently 0to 100,000, wherein n+m≧1; and one L² group is a terminal group (G¹) andthe other L² group is the conjugation tag. In certain instances offormula (II), at least one of B¹, B², M¹ and M² includes -L¹-C¹, whereinL¹ is an optional linker and C¹ is the acceptor chromophore.

As used herein the term “conjugation tag” refers to a group thatincludes a chemoselective functional group (e.g., as described herein)that can covalently link with a compatible functional group of a targetmolecule, after optional activation and/or deprotection. Any convenientconjugation tags may be utilized in the subject polymeric dyes in orderto conjugate the dye to a target molecule of interest. In someembodiments, the conjugation tag includes a terminal functional groupselected from an amino, a carboxylic acid or a derivative thereof, athiol, a hydroxyl, a hydrazine, a hydrazide, a azide, an alkyne and aprotein reactive group (e.g. amino-reactive, thiol-reactive,hydroxyl-reactive, imidazolyl-reactive or guanidinyl-reactive).

Any convenient methods and reagents may be adapted for use in thesubject labelling methods in order to covalently link the conjugationtag to the target molecule. Methods of interest for labelling a target,include but are not limited to, those methods and reagents described byHermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013.The contacting step may be performed in an aqueous solution. In someinstances, the conjugation tag includes an amino functional group andthe target molecule includes an activated ester functional group, suchas a NHS ester or sulfo-NHS ester, or vice versa. In certain instances,the conjugation tag includes a maleimide functional group and the targetmolecule includes a thiol functional group, or vice versa.

Any convenient target molecules may be selected for labelling utilizingthe subject methods. Target molecules of interest include, but are notlimited to, a nucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA orANA molecule, a protein, such as a fusion protein, a modified protein,such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, oracetylated protein, or an antibody, a peptide, an aggregatedbiomolecule, a cell, a small molecule, a vitamin and a drug molecule. Asused herein, the term “a target protein” refers to all members of thetarget family, and fragments thereof. The target protein may be anyprotein of interest, such as a therapeutic or diagnostic target,including but not limited to: hormones, growth factors, receptors,enzymes, cytokines, osteoinductive factors, colony stimulating factorsand immunoglobulins. The term “target protein” is intended to includerecombinant and synthetic molecules, which can be prepared using anyconvenient recombinant expression methods or using any convenientsynthetic methods, or purchased commercially. In some embodiments, thetarget molecule is a specific binding member (e.g., as describedherein). In certain instances, the specific binding member is anantibody. In some instances, the specific binding member is an antibodyfragment or binding derivative thereof. In some case, the antibodyfragment or binding derivative thereof is selected from a Fab fragment,a F(ab′)₂ fragment, a scFv, a diabody and a triabody.

In some cases, the method includes a separating step where the labelledtarget molecule is separated from the reaction mixture, e.g., excessreagents or unlabeled target. A variety of methods may be utilized toseparate a target from a sample, e.g., via immobilization on a support,precipitation, chromatography, and the like.

In some instances, the method further includes detecting and/oranalyzing the labelled target molecule. In some instances, the methodfurther includes fluorescently detecting the labelled target molecule.Any convenient methods may be utilized to detect and/or analyse thelabelled target molecule in conjunction with the subject methods andcompositions. Methods of analyzing a target of interest that find use inthe subject methods, include but are not limited to, flow cytometry,in-situ hybridization, enzyme-linked immunosorbent assays (ELISAs),western blot analysis, magnetic cell separation assays and fluorochromepurification chromatography. Detection methods of interest include butare not limited to fluorescence spectroscopy, nucleic acid sequencing,fluorescence in-situ hybridization (FISH), protein mass spectroscopy,flow cytometry, and the like.

Detection may be achieved directly via a reporter molecule, orindirectly by a secondary detection system. The latter may be based onany one or a combination of several different principles including butnot limited to, antibody labelled anti-species antibody and other formsof immunological or non-immunological bridging and signal amplificationsystems (e.g., biotin-streptavidin technology, protein-A and protein-Gmediated technology, or nucleic acid probe/anti-nucleic acid probes, andthe like). The label used for direct or indirect detection may be anydetectable reported molecule. Suitable reporter molecules may be thoseknown in the field of immunocytochemistry, molecular biology, light,fluorescence, and electron microscopy, cell immunophenotyping, cellsorting, flow cytometry, cell visualization, detection, enumeration,and/or signal output quantification. Labels of interest include, but arenot limited to fluorophores, luminescent labels, metal complexes,radioisotopes, biotin, streptavidin, enzymes, or other detection labelsand combination of labels such as enzymes and a luminogenic substrate.Enzymes of interest and their substrates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and luciferase, and thelike. More than one antibody of specific and/or non-specific naturemight be labelled and used simultaneously or sequentially to enhancetarget detection, identification, and/or analysis. Labels of interestinclude, but are not limited to FITC (fluorescein isothiocyanate) AMCA(7-amino-4-methylcoumarin-3-acetic acid), Alexa Fluor 488, Alexa Fluor594, Alexa Fluor 350, DyLight350, phycoerythrin, allophycocyanin andstains for detecting nuclei such as Hoechst 33342, LDS751, TO-PRO andDAPI.

Systems

Aspects of the invention further include systems for use in practicingthe subject methods and compositions. A sample analysis system mayinclude a flow channel loaded with a sample and a labelled specificbinding member. In some embodiments, the system is a flow cytometricsystem including: a flow cytometer including a flow path; a compositionin the flow path, wherein the composition includes: a sample; and alabelled specific binding member (e.g., as described herein). In someinstances of the system, the labelled specific binding member includes alight harvesting BODIPY unit-comprising multichromophore; and a specificbinding member that specifically binds a target analyte and iscovalently linked to the multichromophore. The multichromophore may be apolymeric dye that is itself fluorescent. The multichromophore may be apolymeric tandem dye. In certain instances, the labelled specificbinding member further includes an acceptor chromophore covalentlylinked to the multichromophore in energy-receiving proximity therewith.In some embodiments, the labelled specific binding member is describedby formula (II):

where: B¹ and B² are each independently a BODIPY unit; each M¹ and eachM² are independently a π conjugated co-monomer; a, b, c, d, e and f areeach independently 0, 1 or 2, wherein b+e≧1; n and m are independently 0to 100,000, wherein n+m≧1; and one L² group is a terminal group (G¹) andthe other L² group is the conjugation tag. In certain instances offormula (II), at least one of B¹, B², M¹ and M² includes -L¹-C¹, whereinL¹ is an optional linker and C¹ is the acceptor chromophore.

In certain embodiments of the system, the composition further includes asecond specific binding member that is support bound and specificallybinds the target analyte. In some cases, the support includes a magneticparticle. As such, in certain instances, the system may also include acontrollable external paramagnetic field configured for application toan assay region of the flow channel.

The sample may include a cell. In some instances, the sample is acell-containing biological sample. In some instances, the sampleincludes a labelled specific binding member specifically bound to atarget cell. In certain instances, the target analyte that isspecifically bound by the specific binding member is a cell surfacemarker of the cell. In certain cases, the cell surface marker isselected from a cell receptor and a cell surface antigen.

In certain aspects, the system may also include a light sourceconfigured to direct light to an assay region of the flow channel. Thesystem may include a detector configured to receive a signal from anassay region of the flow channel, wherein the signal is provided by thefluorescent composition. Optionally further, the sample analysis systemmay include one or more additional detectors and/or light sources forthe detection of one or more additional signals.

In certain aspects, the system may further include computer-basedsystems configured to detect the presence of the fluorescent signal. A“computer-based system” refers to the hardware means, software means,and data storage means used to analyze the information of the presentinvention. The minimum hardware of the computer-based systems of thepresent invention includes a central processing unit (CPU), input means,output means, and data storage means. A skilled artisan can readilyappreciate that any one of the currently available computer-based systemare suitable for use in the subject systems. The data storage means mayinclude any manufacture including a recording of the present informationas described above, or a memory access means that can access such amanufacture.

To “record” data, programming or other information on a computerreadable medium refers to a process for storing information, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats can be used forstorage, e.g., word processing text file, database format, etc.

A “processor” references any hardware and/or software combination thatwill perform the functions required of it. For example, any processorherein may be a programmable digital microprocessor such as available inthe form of an electronic controller, mainframe, server or personalcomputer (desktop or portable). Where the processor is programmable,suitable programming can be communicated from a remote location to theprocessor, or previously saved in a computer program product (such as aportable or fixed computer readable storage medium, whether magnetic,optical or solid state device based). For example, a magnetic medium oroptical disk may carry the programming, and can be read by a suitablereader communicating with each processor at its corresponding station.

In addition to the sensor device and signal processing module, e.g., asdescribed above, systems of the invention may include a number ofadditional components, such as data output devices, e.g., monitorsand/or speakers, data input devices, e.g., interface ports, keyboards,etc., fluid handling components, power sources, etc.

In certain aspects, the system includes a flow cytometer. Flowcytometers of interest include, but are not limited, to those devicesdescribed in U.S. Pat. Nos. 4,704,891; 4,727,029; 4,745,285; 4,867,908;5,342,790; 5,620,842; 5,627,037; 5,701,012; 5,895,922; and 6,287,791;the disclosures of which are herein incorporated by reference.

Other systems may find use in practicing the subject methods. In certainaspects, the system may be a fluorimeter or microscope loaded with asample having a fluorescent composition of any of the embodimentsdiscussed herein. The fluorimeter or microscope may include a lightsource configured to direct light to the assay region of the flowchannel. The fluorimeter or microscope may also include a detectorconfigured to receive a signal from an assay region of the flow channel,wherein the signal is provided by the fluorescent composition.

Kits

Aspects of the invention further include kits for use in practicing thesubject methods and compositions. The compositions of the invention canbe included as reagents in kits either as starting materials or providedfor use in, for example, the methodologies described above.

A kit may include a light harvesting BODIPY unit-comprisingmultichromophore (e.g., as described herein); and one or more componentsselected from a polymeric tandem dye, a fluorophore, a specific bindingmember, a specific binding member conjugate, a support bound specificbinding member, a cell, a support, a biocompatible aqueous elutionbuffer, and instructions for use. In some embodiments of the kit, themultichromophore is covalently linked to a specific binding member. Insome instances, the specific binding member is an antibody. In certaininstances, the specific binding member is an antibody fragment orbinding derivative thereof. In certain cases, the antibody fragment orbinding derivative thereof is selected from a Fab fragment, a F(ab′)2fragment, a scFv, a diabody and a triabody. The multichromophore may bea polymeric dye that is itself fluorescent. The multichromophore may bea polymeric tandem dye. In some cases, the multichromophore furtherincludes an acceptor chromophore covalently linked to themultichromophore in energy-receiving proximity therewith.

In certain embodiments, the kit finds use in evaluating a sample for thepresence of a target analyte, such as an intracellular target. As such,in some instances, the kit includes one or more components suitable forlysing cells. The one or more additional components of the kit may beprovided in separate containers (e.g., separate tubes, bottles, or wellsin a multi-well strip or plate).

In certain aspects, the kit further includes reagents for performing aflow cytometric assay. Reagents of interest include, but are not limitedto, buffers for reconstitution and dilution, buffers for contacting acell sample the multichromophore, wash buffers, control cells, controlbeads, fluorescent beads for flow cytometer calibration and combinationsthereof. The kit may also include one or more cell fixing reagents suchas paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or anycombinations or buffers thereof. Further, the kit may include a cellpermeabilizing reagent, such as methanol, acetone or a detergent (e.g.,triton, NP-40, saponin, tween 20, digitonin, leucoperm, or anycombinations or buffers thereof. Other protein transport inhibitors,cell fixing reagents and cell permeabilizing reagents familiar to theskilled artisan are within the scope of the subject kits.

The compositions of the kit may be provided in a liquid composition,such as any suitable buffer. Alternatively, the compositions of the kitmay be provided in a dry composition (e.g., may be lyophilized), and thekit may optionally include one or more buffers for reconstituting thedry composition. In certain aspects, the kit may include aliquots of thecompositions provided in separate containers (e.g., separate tubes,bottles, or wells in a multi-well strip or plate).

In addition, one or more components may be combined into a singlecontainer, e.g., a glass or plastic vial, tube or bottle. In certaininstances, the kit may further include a container (e.g., such as a box,a bag, an insulated container, a bottle, tube, etc.) in which all of thecomponents (and their separate containers) are present. The kit mayfurther include packaging that is separate from or attached to the kitcontainer and upon which is printed information about the kit, thecomponents of the and/or instructions for use of the kit.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, DVD, portable flash drive, etc., on which theinformation has been recorded. Yet another means that may be present isa website address which may be used via the Internet to access theinformation at a removed site. Any convenient means may be present inthe kits.

Utility

The compositions, methods and systems as described herein may find usein a variety of applications, including diagnostic and researchapplications, in which the labelling detection and/or analysis of atarget of interest is desirable. Such applications include methodologiessuch as cytometry, microscopy, immunoassays (e.g. competitive ornon-competitive), assessment of a free analyte, assessment of receptorbound ligand, and so forth. The compositions, system and methodsdescribed herein may be useful in analysis of any of a number ofsamples, including but not limited to, biological fluids, cell culturesamples, and tissue samples. In certain aspects, the compositions,system and methods described herein may find use in methods whereanalytes are detected in a sample, if present, using fluorescent labels,such as in fluorescent activated cell sorting or analysis, immunoassays,immunostaining, and the like. In certain instances, the compositions andmethods find use in applications where the evaluation of a sample forthe presence of a target analyte is of interest.

In some cases, the methods and compositions find use in any assay formatwhere the detection and/or analysis of a target from a sample is ofinterest, including but not limited to, flow cytometry, in-situhybridization, enzyme-linked immunosorbent assays (ELISAs), western blotanalysis, magnetic cell separation assays and fluorochrome purificationchromatography. In certain instances, the methods and compositions finduse in any application where the fluorescent labelling of a targetmolecule is of interest. The subject compositions may be adapted for usein any convenient applications where pairs of specific binding membersfind use, such as biotin-streptavidin and hapten-anti-hapten antibody.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1

A series of tandem dyes were prepared based on a core Structure 1depicted below including a series of linked fluorophores, DY 633, DY651, DY 682 and DY 752.

FIG. 1 shows absorption and emission of polymeric tandem dyes based oncore Structure 1 attached to a variety of acceptor dyes at the internallinker site (e.g., dyes DY 633, DY 651, DY682 and DY 752). No specificbinding member is attached to these structures.

A second series of tandem dyes were prepared based on a core Structure 1depicted above including a series of linked of acceptor dye fluorophoresat the internal linker site, DY 633, DY 654, DY682, DY 754 and DY 752.FIG. 2 illustrates fluorescence of these polymeric tandem dyes based onStructure 1 with a variety of dye molecules attached at the internallinker site. Absorption for all solutions is 0.04 OD. Note that theemission intensity is significantly higher for polymers with an acceptorchromophore attached relative to the polymer alone. No specific bindingmember is attached to these polymers.

The quantum yield of the tandem pairs is brighter than the core polymeralone. For structure 1 a range of acceptor chromophores were attachedand the resulting tandem pairs were compared spectroscopically to thepolymer without a secondary chromophore. All solutions prepared were atthe same optical density for the excitation wavelength of 562 nm. As canbe seen in FIG. 2, the peak height and peak area for the tandem pairs(peaks 2-6) are all larger than the emission from the underlying polymer(peak 1). In this way extensive prototyping of increasingly brightunderlying polymers is unnecessary and the tandem pairs are limited inbrightness largely due to the quantum yield of the acceptor.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the following.

What is claimed is:
 1. A water soluble polymeric tandem dye comprising:a light harvesting multichromophore that is a conjugated polymercomprising π conjugated BODIPY units; and an acceptor chromophorecovalently linked to the multichromophore in energy-receiving proximitytherewith.
 2. The dye according to claim 1, wherein the multichromophorecomprises a BODIPY-comprising conjugated segment described by formula(I):

wherein: B is a BODIPY unit; M is a π conjugated co-monomer; each L isindependently selected from the group consisting of a terminal group, aπ conjugated segment, a linker and a linked specific binding member; andn is an integer of 2 to 100,000.
 3. The dye according to claim 2,wherein the BODIPY unit is described by the structure:

wherein: R¹, R², R³ and R⁴ are each independently selected from H, analkyl and a substituted alkyl; R⁵ is selected from the group consistingof an alkyl, a substituted alkyl, an aryl, a substituted aryl, aheteroaryl, a substituted heteroaryl, wherein R⁵ is optionallysubstituted with a water solubilizing group; and each R is selected fromthe group consisting of F, OH, H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, alkynyl and substituted alkynyl.
 4. The dyeaccording to claim 1, wherein the multichromophore is described by theformula (II):

wherein: B¹ and B² are each independently a BODIPY unit; each M¹ andeach M² are independently a π conjugated co-monomer; a, b, c, d, e and fare each independently 0, 1 or 2, wherein b+e≧1; n and m areindependently 0 or an integer from 1 to 100,000, wherein n+m≧1; p is aninteger from 1 to 100,000; and each L² is independently selected fromthe group consisting of a terminal group, a π conjugated segment, alinker and a linked specific binding member.
 5. The dye according toclaim 4, wherein at least one of B¹, B², M¹ and M² comprises -L¹-C¹,wherein L¹ is an optional linker and C¹ is the acceptor chromophore. 6.The dye according to claim 4, wherein the dye is described by theformula (III):

wherein each L¹ is an optional linker and each C¹ is an acceptorchromophore.
 7. The dye according to claim 4, wherein the dye isdescribed by the formula (IV):

wherein each L¹ is an optional linker and each C¹ is an acceptorchromophore.
 8. The dye according to claim 4, wherein the dye isdescribed by the formula (V):

wherein each L² is an optional linker and each C¹ is an acceptorchromophore.
 9. The dye according to claim 4, wherein the dye isdescribed by the formula (VI):

wherein each L¹ is an optional linker and each C¹ is an acceptorchromophore.
 10. The dye according to claim 4, wherein B¹ and B² areeach independently described by the following structure:

wherein R⁶ is an aryl or a heteroaryl, optionally substituted with oneor more water solubilizing groups.
 11. The dye according to claim 4,wherein at least one L² is -L³-Z where L³ is a linker and Z is aspecific binding member.
 12. The dye according to claim 4, wherein eachM¹ and M² are each independently described by the following structure:

wherein: each R⁷ is independently selected from the group consisting ofan alkyl, a substituted alkyl, an aralkyl, a substituted aralkyl, a PEGmoiety and -L¹-C¹.
 13. The dye according to claim 4, wherein: M¹ isdescribed by the following structure:

wherein each R⁸ is a substituted aralkyl that comprises a watersolubilizing group; and M² is described by the following structure:

wherein R⁹ is a substituted alkyl that comprises a water solubilizinggroup, and R¹⁹ is -L¹-C¹.
 14. A labelled specific binding member,comprising: a polymeric tandem dye comprising: a water soluble lightharvesting BODIPY unit-comprising multichromophore; an acceptorchromophore covalently linked to the multichromophore inenergy-receiving proximity therewith; and a specific binding membercovalently linked to the multichromophore.
 15. The labelled specificbinding member according to claim 14, wherein the multichromophorecomprises a BODIPY-comprising conjugated segment described by formula(I):

wherein: B is a BODIPY unit; M is a π conjugated co-monomer; each L isindependently selected from the group consisting of a terminal group, aπ conjugated segment, a linker and a linked specific binding member; andn is an integer of 1 to 100,000.
 16. The labelled specific bindingmember according to claim 14, wherein the BODIPY unit is described bythe structure:

wherein: R¹, R², R³ and R⁴ are each independently selected from H, analkyl and a substituted alkyl; R⁵ is selected from the group consistingof an alkyl, a substituted alkyl, an aryl, a substituted aryl, aheteroaryl and a substituted heteroaryl, wherein R⁵ is optionallysubstituted with a water solubilizing group; and each R is selected fromthe group consisting of F, OH, H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,substituted alkoxy, alkynyl and substituted alkynyl.
 17. The labelledspecific binding member according to claim 14, wherein themultichromophore is described by the formula (II):

wherein: B¹ and B² are each independently a BODIPY unit; each M¹ andeach M² are independently a π conjugated co-monomer; a, b, c, d, e and fare each independently 0, 1 or 2, wherein b+e≧1; n and m areindependently 0 to 100,000, wherein n+m≧1; and one L² group is aterminal group (G¹) and the other L² group is a linked specific bindingmember.
 18. The labelled specific binding member according to claim 17,wherein at least one of B¹, B², M¹ and M² comprises -L¹-C¹, wherein L′is an optional linker and C¹ is the acceptor chromophore.
 19. Thelabelled specific binding member according to claim 14, wherein theacceptor chromophore is selected from the group consisting of a cyaninedye, a xanthene dye, a coumarin dye, a thiazine dye and an acridine dye.20. A method of evaluating a sample for the presence of a targetanalyte, the method comprising: (a) contacting the sample with apolymeric tandem dye conjugate that specifically binds the targetanalyte to produce a labelling composition contacted sample, wherein thepolymeric tandem dye conjugate comprises: (i) a light harvesting BODIPYunit-comprising multichromophore; (ii) an acceptor chromophorecovalently linked to the multichromophore in energy-receiving proximitytherewith; and (iii) a specific binding member; and (b) assaying thelabelling composition contacted sample for the presence of a polymerictandem dye conjugate-target analyte binding complex to evaluate whetherthe target analyte is present in the sample.